Method for producing Fmoc-Lys(Ivdde)-OH

Abstract

To provide a method for producing Fmoc-Lys(Ivdde)-OH that simplifies the process, uses mild reaction conditions, is low-cost, and enables industrial production. [Solution] This application provides a method for producing Fmoc-Lys(Ivdde)-OH, comprising the steps of: reacting dimedone and isovaleric acid under the action of a condensing agent to produce an intermediate compound; reacting the product with N-α-tert-butoxycarbonyl-L-lysine under the action of an organic base to obtain an intermediate; reacting the product with an acid to obtain an intermediate; and finally reacting the product with N-(9-fluorenylmethoxycarbonyloxy)succinimide under the action of an inorganic base to obtain Fmoc-Lys(Ivdde)-OH. The method described in this application synthesizes Fmoc-Lys(Ivdde)-OH through a total of four reaction steps: two condensation reactions, a Boc removal reaction, and an Fmoc addition reaction, starting with dimedon. This simplifies the process, makes the operation easy, reduces reaction conditions, lowers the difficulty of industrial production, eliminates the need for valuable raw materials, helps reduce production costs, and yields a final product of high purity and high yield.

Description

Technical Field

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

Background Art

[0002] Fmoc-Lys(Ivdde)-OH is an intermediate commonly used in the synthesis of peptides such as Semaglutide and Tirzepatide. Fmoc-Lys(Ivdde)-OH has the Chinese name: N-Fluorenylmethyloxycarbonyl-N’-[1-(4,4-dimethyl-2,6-dioxocyclohexylmethylene)-3-methylbutyl]-L-lysine, molecular formula: C 34 H 42 N2O6, CAS number: 204777-78-6, and is represented by the following structural formula.

Chemical

[0003] In the conventional technology (Chhabra, Siri Ram et al., Tetrahedron Letters (1998), 39(12), 1603-1606), as shown in the following reaction process, a method for producing Fmoc-Lys(Ivdde)-OH has been proposed, which includes refluxing Fmoc-Lys, Ivdde-OH and trifluoroacetic acid in ethanol for 60 hours and performing post-treatment to obtain Fmoc-Lys(Ivdde)-OH.

Chemical

[0004] However, in the above reaction process, trifluoroacetic acid has strong corrosiveness and the reaction is carried out at high temperature for a long time, so the safety risk is large and it is not suitable for large-scale production.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therefore, the present invention aims to provide a method for producing Fmoc-Lys(Ivdde)-OH that simplifies the process, requires mild reaction conditions, is cost-effective, and can be industrially produced. [Means for solving the problem]

[0006] According to this application, a method for producing Fmoc-Lys(Ivdde)-OH, [ka] Step a) involves reacting dimedone and isovaleric acid under the action of a condensing agent to produce the intermediate compound Ivdde-OH shown in formula (3) above, [ka] Step b) involves reacting the intermediate compound Ivdde-OH shown in formula (3) with N-α-tert-butoxycarbonyl-L-lysine under the action of an organic base to obtain the intermediate Boc-Lys(Ivdde)-OH shown in formula (5), [ka] Step c) involves reacting the intermediate Boc-Lys(Ivdde)-OH shown in formula (5) with an acid to obtain the intermediate Lys(Ivdde)-OH shown in formula (6), [ka] A method for producing Fmoc-Lys(Ivdde)-OH is provided, which includes step d) reacting the intermediate Lys(Ivdde)-OH shown in formula (6) with N-(9-fluorenylmethoxycarbonyloxy)succinimide under the action of an inorganic base to obtain Fmoc-Lys(Ivdde)-OH shown in formula (8).

[0007] In step a) of some specific embodiments, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or dicyclohexylcarbodiimide, and the molar ratio of dimedone, isovaleric acid, and condensing agent is 1:(0.8~1.3):(1~1.5).

[0008] In step a) of some specific embodiments, the reaction medium is dichloromethane, acetone, or tetrahydrofuran, and the reaction is carried out at room temperature for 12 to 16 hours.

[0009] In step b) of some specific embodiments, the organic base is triethylamine or N,N-diisopropylethylamine, and the molar ratio of N-α-tert-butoxycarbonyl-L-lysine, Ivdde-OH, and the organic base is 1:(1~1.5):(1.1~2.0).

[0010] In step b) of some specific embodiments, the reaction medium is acetonitrile, methanol, or ethanol, and the reaction is carried out at a temperature of 65°C to 75°C for 12 to 14 hours.

[0011] In step c) of some specific embodiments, the acid is hydrochloric acid, and the molar ratio of Boc-Lys(Ivdde)-OH to the acid is 1:(5~8).

[0012] In step c) of some specific embodiments, the reaction is carried out at room temperature for 6 to 8 hours.

[0013] In step d) of some specific embodiments, the inorganic base is selected from the group consisting of sodium carbonate, sodium bicarbonate, and potassium carbonate.

[0014] In step d) in some specific embodiments, the molar ratio of Lys(Ivdde)-OH, N-(9-fluorenylmethoxycarbonyloxy)succinimide, and inorganic base is 1:(0.7 - 1.0):(1.2 - 1.5).

[0015] In step d) in some specific embodiments, the medium of the reaction is ethyl acetate, acetone, or tetrahydrofuran, and the reaction is carried out at a temperature of 25°C - 30°C for 3 - 4 hours.

[0016] According to the present application, there is provided a method for producing Fmoc-Lys(Ivdde)-OH, which includes step a) of reacting dimedone and isovaleric acid under the action of a condensing agent to produce the intermediate compound Ivdde-OH shown in formula (3); step b) of reacting the intermediate compound Ivdde-OH shown in formula (3) and N-α-tert-butoxycarbonyl-L-lysine under the action of an organic base to obtain the intermediate Boc-Lys(Ivdde)-OH shown in formula (5); step c) of reacting the intermediate Boc-Lys(Ivdde)-OH shown in formula (5) with an acid to obtain the intermediate Lys(Ivdde)-OH shown in formula (6); and step d) of reacting the intermediate Lys(Ivdde)-OH shown in formula (6) with N-(9-fluorenylmethoxycarbonyloxy)succinimide under the action of an inorganic base to obtain Fmoc-Lys(Ivdde)-OH shown in formula (8). The method according to the present application uses dimedone as a starting material and synthesizes Fmoc-Lys(Ivdde)-OH through a total of four steps of reactions, namely two condensation reactions, a Boc removal reaction, and an Fmoc addition reaction. Therefore, the process is simplified, the operation is convenient, the reaction conditions are mild, the difficulty of industrial production is reduced, precious raw materials are not required, which helps to reduce production costs, and has the advantages that Fmoc-Lys(Ivdde)-OH can be obtained with high purity and high yield. From the experimental results, it was clearly shown that the Fmoc-Lys(Ivdde)-OH produced by the method according to the present application reached a purity of 99% or more and a yield of 75% or more (calculated based on Boc-Lys).

Brief Description of the Drawings

[0017] [Figure 1] The HPLC chromatogram of Fmoc-Lys(Ivdde)-OH manufactured in Example 2 of this application. [Figure 2] The infrared spectrum of Fmoc-Lys(Ivdde)-OH manufactured in Example 2 of this application. [Figure 3] The nuclear magnetic resonance spectrum of Fmoc-Lys(Ivdde)-OH manufactured in Example 2 of this application.

Mode for Carrying Out the Invention

[0018] The technical solution of the present invention will be clearly and completely described below with reference to examples. Of course, the examples described herein are only a part of the examples of the present invention, not all of them. All other examples that can be obtained by those skilled in the art without making inventive efforts shall be included within the protection scope of the present invention.

[0019] In the description of the present invention, it should be understood that terms such as "center", "vertical direction", "horizontal direction", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc. are based on the positions or locations shown in the accompanying drawings. These terms are only intended to facilitate the description of the present invention and simplify the description, and do not explicitly or implicitly imply that the devices or components mentioned herein necessarily have a specific direction and are configured or operated in a specific direction, so they should not be construed as limiting the present invention.

[0020] Furthermore, the terms “first” and “second” are used solely for descriptive purposes and should not be understood as expressing or implying relative importance, or implicitly meaning the number of technical features described. Thus, features designated as “first” or “second” may explicitly or implicitly include one or more of the aforementioned features. In the description of this invention, “multiple” means two or more unless specifically and clearly limited. Moreover, the terms “attachment,” “joining,” and “connection” should be understood in a broad sense. For example, they may be fixed connections, detachable connections, or integrated connections. They may also be mechanical connections or electrical connections. They may also be direct connections, indirect connections via an intermediate medium, or communication between two elements. To those skilled in the art, the specific meanings of the above terms in this invention can be understood depending on the specific context.

[0021] According to this application, a method for producing Fmoc-Lys(Ivdde)-OH, [ka] Step a) involves reacting dimedone and isovaleric acid under the action of a condensing agent to produce the intermediate compound Ivdde-OH shown in formula (3) above, [ka] Step b) involves reacting the intermediate compound Ivdde-OH shown in formula (3) with N-α-tert-butoxycarbonyl-L-lysine under the action of an organic base to obtain the intermediate Boc-Lys(Ivdde)-OH shown in formula (5), [ka] Step c) involves reacting the intermediate Boc-Lys(Ivdde)-OH shown in formula (5) with an acid to obtain the intermediate Lys(Ivdde)-OH shown in formula (6), [ka] A method for producing Fmoc-Lys(Ivdde)-OH is provided, which includes step d) reacting the intermediate Lys(Ivdde)-OH shown in formula (6) with N-(9-fluorenylmethoxycarbonyloxy)succinimide under the action of an inorganic base to obtain Fmoc-Lys(Ivdde)-OH shown in formula (8).

[0022] This invention offers several advantages: it allows for the synthesis of Fmoc-Lys(Ivdde)-OH starting from dimedone through a total of four reaction steps—two condensation reactions, a Boc removal reaction, and an Fmoc addition reaction—simplifies the process, simplifies the operation, requires mild reaction conditions, reduces the difficulty of industrial production, eliminates the need for valuable raw materials, helps reduce production costs, and allows for the acquisition of Fmoc-Lys(Ivdde)-OH in high purity and high yield.

[0023] In this application, as shown in the reaction process below, dimedone and isovaleric acid are used as raw materials and reacted under the action of a condensing agent to produce the intermediate compound Ivdde-OH shown in formula (3). [ka]

[0024] Dimedone has the structure represented by formula (1), and the source of its supply is not particularly limited in this application. Isovaleric acid has the structure represented by formula (2), and the source of its supply is not particularly limited in this application.

[0025] In some specific embodiments, the condensing agent in step a) may be, but is not limited to, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) or dicyclohexylcarbodiimide (DCC), and may be one or more of these. If the condensing agent is a combination of multiple substances, this application does not particularly limit the mixing ratio of these substances. In some specific embodiments, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) or dicyclohexylcarbodiimide (DCC).

[0026] In some specific embodiments, the reaction medium in step a) is an organic solvent. In some specific embodiments, the organic solvent is ethyl acetate, acetone, or tetrahydrofuran.

[0027] In some specific embodiments, the molar ratio of dimedone, isovaleric acid, and the condensing agent is 1:(0.8~1.3):(1~1.5), preferably 1:1:1.1.

[0028] Specifically, in this application, first, dimedone, isovaleric acid, and an organic solvent are uniformly mixed, and then a condensing agent is added and the mixture is reacted to obtain the intermediate compound Ivdde-OH shown in formula (3). In some specific embodiments, the reaction is carried out by adding the condensing agent at a temperature of 15°C to 20°C. In some specific embodiments, the reaction temperature is room temperature, for example, in the range of 25°C to 35°C, preferably in the range of 28°C to 32°C, and the reaction time is in the range of 12 to 16 hours, preferably in the range of 13 to 15 hours.

[0029] After the reaction is complete, the reaction product is post-processed. Specifically, this process includes adding water to the reaction system, allowing it to stand to separate into layers, and then recovering the organic layer, and drying and concentrating the organic layer to obtain the intermediate compound Ivdde-OH shown in formula (3).

[0030] This application is not particularly limited by the drying method. For example, it can be dried with sodium sulfate. After drying, concentration yields the intermediate compound Ivdde-OH shown in formula (3). This can be used directly in the next step without purification.

[0031] As shown in the following reaction process, the intermediate compound Ivdde-OH shown in formula (3) is reacted with N-α-tert-butoxycarbonyl-L-lysine under the action of an organic base to obtain the intermediate Boc-Lys(Ivdde)-OH shown in formula (5). [ka]

[0032] N-α-tert-butoxycarbonyl-L-lysine (Boc-Lys) has the structure represented by formula (4), and there are no particular restrictions on its source in this application; it can be purchased from the market.

[0033] In some specific embodiments, the organic base may be, but is not limited to, triethylamine and N,N-diisopropylethylamine, and may be one or more of these. When the organic base is two or more of these, the proportion of each substance is not particularly limited in this application. In some specific embodiments, the organic base is triethylamine or N,N-diisopropylethylamine.

[0034] In some specific embodiments, in step b), the reaction medium is an organic solvent. In some specific embodiments, the organic solvent is acetonitrile, methanol, or ethanol.

[0035] In some specific embodiments, the molar ratio of Boc-Lys, Ivdde-OH, and organic base is 1:(1~1.5):(1.1~2.0), preferably 1:1.1:1.5.

[0036] Specifically, in this application, Boc-Lys, Ivdde-OH, and an organic base are homogeneously mixed with an organic solvent, the temperature is raised, and the reaction is carried out under reflux to obtain the intermediate Boc-Lys(Ivdde)-OH shown in formula (5). In some specific embodiments, the reaction is carried out at a temperature of 65°C to 75°C for 12 to 14 hours. In some specific embodiments, the reaction is carried out under stirring conditions.

[0037] After the reaction is complete, the reaction product is post-treated. Specifically, this includes the steps of: concentrating the reaction system to dryness, cooling, adding water, an organic solvent, and an acid, allowing it to stand and separate, and recovering the organic layer; and washing, drying, concentrating, crystallizing, filtering, and re-drying the organic layer to obtain the intermediate Boc-Lys(Ivdde)-OH shown in formula (5).

[0038] In this application, first, the reaction system is concentrated to dryness, preferably cooled to room temperature, and then water, an organic solvent, and an acid are added. Here, ethyl acetate may be used as the organic solvent, and the pH of the reaction system is adjusted to 4-5 by adding the acid. In this application, the type of acid is not particularly limited and may be citric acid. After static separation, in this application, the method of washing the recovered organic layer is not particularly limited and can be washed three times with saturated sodium chloride, for example. In this application, the drying method is not particularly limited and can be dried with sodium sulfate, for example. After drying, it is filtered, and the obtained filtrate is concentrated until a large amount of solid matter precipitates, cooled again, and then crystallized. In this application, it is preferable to add a solvent such as petroleum ether and crystallize for 1-3 hours. The obtained product is filtered and re-dried to obtain the intermediate Boc-Lys(Ivdde)-OH shown in formula (5). In this application, the drying method is not particularly limited and can be dried at 50°C-55°C.

[0039] As shown in the following reaction process, the intermediate Boc-Lys(Ivdde)-OH shown in formula (5) is reacted with an acid to obtain the intermediate Lys(Ivdde)-OH shown in formula (6). [ka]

[0040] In some specific embodiments, the acid is hydrochloric acid and functions to remove the Boc protecting group.

[0041] In some specific embodiments, the molar ratio of Boc-Lys(Ivdde)-OH to the acid is 1:(5~8), preferably 1:7.

[0042] In some specific embodiments, the reaction medium is an organic solvent, such as tetrahydrofuran, but is not limited to these.

[0043] Specifically, in this application, an organic solvent and an acid are mixed and then reacted with the intermediate Boc-Lys(Ivdde)-OH shown in formula (5) to obtain the intermediate Lys(Ivdde)-OH shown in formula (6). In some specific embodiments, the reaction is carried out at room temperature, for example, 20°C to 25°C, for 6 to 8 hours.

[0044] After the reaction is complete, a base is added to the resulting reaction system to adjust the pH to 6-7. After allowing the system to stand and separate into layers, the aqueous layer is collected to obtain the intermediate Lys(Ivdde)-OH shown in equation (6). The obtained intermediate can proceed to the next step without further processing.

[0045] As shown in the following reaction process, the intermediate Lys(Ivdde)-OH shown in formula (6) is reacted with N-(9-fluorenylmethoxycarbonyloxy)succinimide under the action of an inorganic base to obtain Fmoc-Lys(Ivdde)-OH shown in formula (8). [ka]

[0046] N-(9-Fluorenylmethoxycarbonyloxy)succinimide (Fmoc-Osu) has the structure represented by formula (7), and the source thereof is not particularly limited in this application.

[0047] In some specific embodiments, the inorganic base may include, but is not limited to, sodium carbonate, sodium bicarbonate, potassium carbonate, and may be one or more of these. When the inorganic base is two or more of these, this application does not particularly limit the proportion of each substance.

[0048] In some specific embodiments, the reaction is carried out in an organic solvent, which includes, but is not limited to, ethyl acetate, acetone, and tetrahydrofuran.

[0049] In some specific embodiments, the molar ratio of Lys(Ivdde)-OH, Fmoc-Osu, and the inorganic base is 1:(0.7~1.0):(1.2~1.5), preferably 1:0.8:1.4.

[0050] Specifically, in this application, an aqueous solution of Lys(Ivdde)-OH, an organic solvent, and an inorganic base are uniformly mixed, and then Fmoc-Osu is added and reacted to obtain Fmoc-Lys(Ivdde)-OH shown in formula (8). In some specific embodiments, it is preferable to add the Fmoc-Osu at a temperature of 25°C to 30°C, and the reaction temperature is preferably 25°C to 30°C. If the reaction temperature is too low, the reaction time will be long, and if the reaction temperature is too high, impurities will increase and become difficult to remove. In some specific embodiments, the reaction time is set to 3 to 4 hours. In some specific embodiments, the reaction is carried out under stirring conditions.

[0051] It is preferable to perform post-treatment after the reaction is complete. 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 obtaining Fmoc-Lys(Ivdde)-OH shown in formula (8) by washing, drying, concentrating, crystallizing, centrifuging, and drying the organic layer.

[0052] In this application, the acid is not particularly limited and may be concentrated hydrochloric acid. In this application, the washing method is not particularly limited and can be washed three times with saturated sodium chloride, for example. In this application, the drying method is not particularly limited and can be dried with sodium sulfate, for example. In this application, the crystallization method is not particularly limited and can be cooled to 20°C to 25°C and crystallized by adding an organic solvent, for example. Here, the organic solvent can be petroleum ether, but is not limited thereto. Crystallization is preferably carried out for 1 to 3 hours. In this application, the drying method after centrifugation is not particularly limited and can be dried at a temperature of 50°C to 55°C, for example.

[0053] After the reaction was complete, HPLC analysis, infrared analysis, and nuclear magnetic resonance analysis were performed on the obtained solid. The results revealed that the obtained solid was Fmoc-Lys(Ivdde)-OH, with a purity of over 99% and a yield of over 75% (calculated using Boc-Lys). [Examples]

[0054] The method for producing Fmoc-Lys(Ivdde)-OH according to this application will be described in detail below with reference to examples.

[0055] [Example 1] (1) Preparation of Ivdde-OH: 650 g of dichloromethane was placed in a 1 L three-necked flask, and while stirring, 100 g of dimedone (shown in formula (1) below) and 73 g of isovaleric acid (shown in formula (2) below) were added and stirred until completely dissolved. Next, 150 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl) was added, and the temperature was controlled to 15°C to 20°C. After the addition of the raw materials was complete, the reaction was allowed to proceed for 13 hours while controlling the temperature to 25°C to 30°C, and the complete reaction of the raw material dimedone was confirmed by TLC. 200 g of water was added to the system and stirred until clarified. After layer separation occurred upon standing, the dichloromethane layer was collected. Sodium sulfate was added to the dichloromethane layer and dried, then filtered, and the filtrate was concentrated to dryness to obtain 121 g of an oily substance, Ivdde-OH (shown in formula (3) below), in 81.2% yield. This oily substance was used directly in the next step without detection or purification. The reaction process in step (1) is shown below: [ka]

[0056] (2) Preparation of Boc-Lys(Ivdde)-OH: 726 g of methanol was placed in a 2 L three-necked flask, and 121 g of N-α-tert-butoxycarbonyl-L-lysine (Boc-Lys) shown in formula (4), 121 g of Ivdde-OH prepared in step (1), and 95 g of N,N-diisopropylethylamine were added. The internal temperature was raised to 70°C to 75°C, and the reaction was allowed to proceed for 13 hours. Afterward, the complete reaction of the starting material Boc-Lys was confirmed by TLC. The methanol in the reaction system was concentrated to dryness and cooled to 20°C to 30°C. 200 g of ethyl acetate and 200 g of water were added to the reaction solution, and the pH of the system was acidified to 4 to 5 with citric acid. After separation of layers by standing, the ethyl acetate layer was collected and washed three times with saturated sodium chloride. After washing, sodium sulfate was added and the mixture was dried, and the mixture was filtered and concentrated until a large amount of solid matter precipitated. Next, the temperature was cooled to 20°C to 25°C, 200g of petroleum ether was added and crystallized for 2 hours. After filtration, the mixture was dried at 50°C to 55°C to obtain 198g of a solid, which was the intermediate Boc-Lys(Ivdde)-OH. The yield was 89%, and the HPLC purity was 99.18%. The reaction process in step (2) is shown below. [ka]

[0057] (3) Preparation of Lys(Ivdde)-OH: 560 g of tetrahydrofuran and 612 g of 6N hydrochloric acid were added to a 2 L three-necked flask. While stirring, 198 g of Boc-Lys(Ivdde)-OH prepared in step (2) was added, and the reaction was carried out for 6 hours while controlling the temperature to 20°C to 25°C. The completion of the Boc-Lys(Ivdde)-OH reaction was confirmed by TLC. Sodium carbonate was slowly added to the reaction system to adjust the pH to 6 to 7. After pH adjustment, the system was allowed to stand and separate layers occurred. The aqueous layer, which is an aqueous solution of Lys(Ivdde)-OH as shown in formula (6), was collected. This aqueous solution was used directly in the next step. The reaction process in step (3) is shown below. [ka]

[0058] (4) Preparation of Fmoc-Lys(Ivdde)-OH: The Lys(Ivdde)-OH aqueous solution prepared in step (3) was added to a 2 L three-necked flask, and 750 g of ethyl acetate and 118 g of sodium carbonate were added. After homogeneous stirring, 130 g of N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-Osu) was added while controlling the temperature to 25°C to 30°C, and the mixture was reacted for 3 hours. Complete reaction of Fmoc-Osu was confirmed by TLC. The pH of the system was adjusted to 2 to 3 with concentrated hydrochloric acid, and after separation of layers occurred by standing, the ethyl acetate layer was collected and washed three times with saturated sodium chloride. After washing, sodium sulfate was added and the mixture was dried, filtered, and concentrated until a large amount of solid precipitated. The mixture was cooled to 20°C to 25°C, and 350 g of petroleum ether was added and crystallized for 2 hours. Next, the mixture was filtered and dried at 50°C to 55°C to obtain 182 g of solid, which was the intermediate Fmoc-Lys(Ivdde)-OH. The yield was 82.18%, and the HPLC purity was 99.69%. The reaction process in step (4) is shown below. [ka]

[0059] [Example 2] (1) Preparation of Ivdde-OH: 150 kg of dichloromethane was placed in a 500 L reaction vessel, and 23 kg of dimedone and 16.8 kg of isovaleric acid were added while stirring, and the mixture was stirred until completely dissolved. Next, 37.1 kg of dicyclohexylcarbodiimide (DCC) was added, and the temperature was controlled to 15°C to 20°C. After the addition of the raw materials was complete, the reaction was carried out for 14 hours while controlling the temperature to 25°C to 30°C, and the complete reaction of the raw material dimedone was confirmed by TLC. Insoluble material (DCU) in the system was filtered out, and the filtrate was collected. The filtrate was directly concentrated to dryness, and 31 kg of oily substance was obtained in 84.23% yield. This oily substance was used directly in the next step without detection or purification.

[0060] (2) Preparation of Boc-Lys(Ivdde)-OH: 186 kg of ethanol was placed in a clean, anhydrous 500 L reaction vessel. While stirring, 31 kg of Boc-Lys, 31 kg of Ivdde-OH produced in step (1), and 19 kg of triethylamine were added. The internal temperature was raised to 70°C to 75°C and the reaction was carried out for 13 hours. The complete reaction of the starting material, Boc-Lys, was confirmed by TLC. The ethanol in the reaction system was concentrated to dryness and cooled to 20°C to 30°C. 100 kg of ethyl acetate and 100 kg of water were added to the reaction solution, and the pH of the system was acidified to 4 to 5 with citric acid. Next, after separation of layers occurred by standing, the ethyl acetate layer was collected and washed three times with saturated sodium chloride. After washing, sodium sulfate was added, the mixture was dried, filtered, and concentrated until a large amount of solid matter precipitated. The mixture was cooled to 20-25°C, 100 kg of petroleum ether was added, and crystallization was carried out for 2 hours. The centrifuged solid was dried at 50-55°C to obtain 53 kg of solid, which is the intermediate Boc-Lys(Ivdde)-OH. Referring to Figure 1, Figure 1 is the HPLC spectrum of the intermediate Boc-Lys(Ivdde)-OH produced in Example 2 of this application. The yield was 93%, and the HPLC purity was 99.28%. The HPLC data is shown in the table below. [Table 0]

[0061] (3) Preparation of Lys(Ivdde)-OH: 150 kg of tetrahydrofuran and 164 kg of 6N hydrochloric acid were added to a clean 500 L reaction vessel. While stirring, 53 kg of Boc-Lys(Ivdde)-OH produced in step (2) was added, and the reaction was carried out for 7 hours while controlling the temperature to 20°C to 25°C. The complete reaction of the starting material Boc-Lys(Ivdde)-OH was confirmed by TLC. Sodium carbonate was slowly added to the system to adjust the pH to 6 to 7. After pH adjustment, the mixture was allowed to stand and separate layers occurred, after which the aqueous layer, which was an aqueous solution of Lys(Ivdde)-OH, was collected. This aqueous solution was then used directly to proceed to the next step.

[0062] (4) Preparation of Fmoc-Lys(Ivdde)-OH: The Lys(Ivdde)-OH aqueous solution prepared in step (3) was added to a 1000L reaction vessel, followed by 200kg of ethyl acetate and 31.5kg of sodium carbonate. After homogeneous stirring, the temperature was controlled to 25°C-30°C, 35kg of Fmoc-Osu was added, and the mixture was reacted for 3 hours. Complete reaction of Fmoc-Osu was confirmed by TLC. The pH of the system was adjusted to 2-3 with concentrated hydrochloric acid, and after separation of layers occurred, the ethyl acetate layer was collected and washed three times with saturated sodium chloride. After washing, sodium sulfate was added to dry the mixture, and it was filtered and concentrated until a large amount of solid precipitated. The mixture was cooled to 20°C-25°C, 100kg of petroleum ether was added, and crystallization was carried out for 2 hours. The mixture was centrifuged and dried at 50°C-55°C to obtain 50kg of solid, which was the intermediate Fmoc-Lys(Ivdde)-OH. The yield was 83.9%, and the HPLC purity was 99.70%. Referring to Figures 1, 2, and 3, Figure 1 is the HPLC spectrum of Fmoc-Lys(Ivdde)-OH produced in Example 2 of this application, Figure 2 is the infrared spectrum of Fmoc-Lys(Ivdde)-OH produced in Example 2 of this application, and Figure 3 is the nuclear magnetic resonance spectrum of Fmoc-Lys(Ivdde)-OH produced in Example 2 of this application.

[0063] [Example 3] Based on the manufacturing method of step (1) of Example 1, only the type of condensing agent was changed, and the other manufacturing steps and process parameters were the same as in step (1) of Example 1. The yield and purity in step (1) are as shown in Table 1 below.

[0064] [Table 1]

[0065] [Example 4] Based on the manufacturing method of step (2) of Example 1, the only difference was the type of organic solvent; all other manufacturing steps and process parameters were the same as in step (2) of Example 1. The yield and purity in step (2) are as shown in Table 2 below.

[0066] [Table 2]

[0067] [Examples 5-6, Comparative Examples 1-2] Based on the manufacturing method of step (2) of Example 1, only the reaction temperature was changed, and the other manufacturing steps and process parameters were the same as in step (2) of Example 1. The yield and purity in step (2) are as shown in Table 3 below.

[0068] [Table 3]

[0069] [Examples 7-8] Based on the manufacturing method in step (4) of Example 1, the only difference was the type of inorganic base; all other manufacturing steps and process parameters were the same as in step (4) of Example 1. The yield and purity in step (4) are shown in Table 4 below.

[0070] [Table 4]

[0071] [Examples 9-10] Based on the manufacturing method in step (4) of Example 1, the only difference was the type of organic solvent; all other manufacturing steps and process parameters were the same as in step (4) of Example 1. The yield and purity in step (4) are as shown in Table 5 below.

[0072] [Table 5]

[0073] As described above, the present invention provides a method for producing Fmoc-Lys(Ivdde)-OH that is suitable for scale-up. This synthesis method requires simple equipment, has high reaction efficiency, low production costs, and the final product has a purity of 99.0% or more and a yield of 75% or more (calculated using Boc-Lys).

[0074] 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 dimedone and isovaleric acid under the action of a condensing agent to produce the intermediate compound Ivdde-OH shown in formula (3) above, 【Chemistry 2】 Step b) involves reacting the intermediate compound Ivdde-OH shown in formula (3) with N-α-tert-butoxycarbonyl-L-lysine under the action of an organic base to obtain the intermediate Boc-Lys(Ivdde)-OH shown in formula (5) above, 【Transformation 3】 Step c) involves reacting the intermediate Boc-Lys(Ivdde)-OH shown in formula (5) with an acid to obtain the intermediate Lys(Ivdde)-OH shown in formula (6) above, 【Chemistry 4】 Step d) involves reacting the intermediate Lys(IVdde)-OH shown in formula (6) with N-(9-fluorenylmethoxycarbonyloxy)succinimide under the action of an inorganic base to obtain Fmoc-Lys(IVdde)-OH shown in formula (8) above, A method for producing Fmoc-Lys(IVdde)-OH, characterized by containing the following:

2. In step a), the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and / or dicyclohexylcarbodiimide, The manufacturing method according to claim 1, characterized in that the molar ratio of dimedone, isovaleric acid, and condensing agent is 1:(0.8-1.3):(1-1.5).

3. In step a), the reaction medium is dichloromethane, acetone, or tetrahydrofuran. The above reaction is carried out at room temperature for 12 to 16 hours. The manufacturing method according to claim 2, characterized in that it

4. In step b), the organic base is triethylamine and / or N,N-diisopropylethylamine. The molar ratio of N-α-tert-butoxycarbonyl-L-lysine, Ivdde-OH, and the organic base is 1:(1-1.5):(1.1-2.0). The manufacturing method according to claim 1, characterized in that it

5. In step b), the reaction medium is acetonitrile, methanol, or ethanol. The above reaction is carried out at a temperature of 65°C to 75°C for 12 to 14 hours. The manufacturing method according to feature 4.

6. In step c), the acid is hydrochloric acid. The molar ratio of Boc-Lys(Ivdde)-OH to the acid is 1:(5-8). The manufacturing method according to claim 1, characterized in that it

7. The manufacturing method according to claim 6, characterized in that in step c), the reaction is carried out at room temperature for 6 to 8 hours.

8. The manufacturing method according to claim 1, characterized in that in step d), the inorganic base is sodium carbonate, sodium bicarbonate, and / or potassium carbonate.

9. The manufacturing method according to claim 8, characterized in that in step d), the molar ratio of Lys(IVdde)-OH, N-(9-fluorenylmethoxycarbonyloxy)succinimide, and inorganic base is 1:(0.7 to 1.0):(1.2 to 1.5).

10. In step d), the reaction medium is ethyl acetate, acetone, or tetrahydrofuran. The above reaction is carried out at a temperature of 25°C to 30°C for 3 to 4 hours. The manufacturing method according to claim 8.

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