Preparation method of Fmoc-Lys (Ivdde)-OH

Abstract

The invention provides a preparation method of Fmoc-Lys (Ivdde)-OH, which comprises the following steps: reacting dimedone with isovaleric acid under the action of a condensing agent to generate an intermediate compound, then reacting with N-alpha-tert-butoxycarbonyl-L-lysine under the action of organic alkali to obtain an intermediate, then reacting with acid to obtain an intermediate, and finally, separating and purifying the intermediate to obtain the Fmoc-Lys (Ivdde)-OH. Finally, under the action of inorganic alkali, the Fmoc-Lys (Ivdde)-OH reacts with fluorenylmethoxycarbonyl succinimide, and Fmoc-Lys (Ivdde)-OH is obtained. According to the method provided by the invention, the diketone is taken as a starting raw material, Fmoc-Lys (Ivdde)-OH is synthesized through four steps of reaction including two-step condensation reaction, Boc removal reaction and Fmoc feeding reaction, 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-Lys(Ivdde)-OH. Background Art

[0002] Fmoc-Lys(Ivdde)-OH is a commonly used intermediate in the synthesis of peptides such as semaglutide and telportide. The Chinese name of Fmoc-Lys(Ivdde)-OH is N-fluorenylmethoxycarbonyl-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylmethylene)-3-methylbutyl]-L-lysine, and its molecular formula is C 34 H 42 N2O6, CAS No. 204777-78-6, has the following structural formula:

[0003]

[0004] The prior art (By: Chhabra, Siri Ram; et al Tetrahedron Letters (1998), 39 (12), 1603-1606) proposed a method for preparing Fmoc-Lys (Ivdde) -OH, which uses Fmoc-Lys, Ivdde-OH and trifluoroacetic acid to reflux in ethanol for 60 hours and obtain Fmoc-Lys (Ivdde) -OH through post-treatment. The reaction process is as follows:

[0005]

[0006] However, in the above reaction process, trifluoroacetic acid is highly corrosive, and the reaction is carried out at high temperature for a long time, which poses a great safety risk and is not conducive to large-scale production. Summary of the invention

[0007] In view of this, the object of the present invention is to provide a method for preparing Fmoc-Lys(Ivdde)-OH. The preparation method provided by the present invention has simple process, mild reaction conditions, low cost, and can realize industrial production.

[0008] The present application provides a method for preparing Fmoc-Lys(Ivdde)-OH, comprising the following steps:

[0009] a) dimedone and isovaleric acid react under the action of a condensing agent to generate an intermediate compound Ivdde-OH shown in formula (3);

[0010]

[0011] b) the intermediate compound Ivdde-OH represented by formula (3) reacts with N-alpha-tert-butoxycarbonyl-L-lysine under the action of an organic base to obtain the intermediate Boc-Lys(Ivdde)-OH represented by formula (5);

[0012]

[0013] c) reacting the intermediate Boc-Lys(Ivdde)-OH represented by formula (5) with an acid to obtain the intermediate Lys(Ivdde)-OH represented by formula (6);

[0014]

[0015] d) reacting the intermediate Lys(Ivdde)-OH represented by formula (6) with fluorenylmethoxycarbonyl succinimide under the action of an inorganic base to obtain Fmoc-Lys(Ivdde)-OH represented by formula (8);

[0016]

[0017] In some specific implementations, in step a), the condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or dicyclohexylcarbodiimide;

[0018] The molar ratio of dimedone, isovaleric acid and condensing agent is 1:(0.8-1.3):(1-1.5).

[0019] In some specific implementations, in step a), the reaction medium is dichloromethane, acetone or tetrahydrofuran;

[0020] The reaction temperature is room temperature and the reaction time is 12 h to 16 h.

[0021] In some specific implementations, in step b), the organic base is triethylamine or N,N-diisopropylethylamine;

[0022] The molar ratio of N-alpha-tert-butoxycarbonyl-L-lysine, Ivdde-OH and the organic base is 1:(1-1.5):(1.1-2.0).

[0023] In some specific implementations, in step b), the reaction medium is acetonitrile, methanol or ethanol;

[0024] The reaction temperature is 65°C to 75°C, and the reaction time is 12h to 14h.

[0025] In some specific implementations, in step c), the acid is hydrochloric acid;

[0026] The molar ratio of the Boc-Lys(Ivdde)-OH to the acid is 1:(5-8).

[0027] In some specific implementations, in step c), the reaction temperature is room temperature and the reaction time is 6 h to 8 h.

[0028] In some specific implementations, in step d), the inorganic base is sodium carbonate, sodium bicarbonate or potassium carbonate.

[0029] In some specific implementations, in step d), the molar ratio of Lys(Ivdde)-OH, fluorenylmethoxycarbonyl succinimide and the inorganic base is 1:(0.7-1.0):(1.2-1.5).

[0030] In some specific implementations, in step d), the reaction medium is ethyl acetate, acetone or tetrahydrofuran;

[0031] The reaction temperature is 25°C to 30°C, and the reaction time is 3h to 4h.

[0032] The present application provides a method for preparing Fmoc-Lys(Ivdde)-OH, comprising the following steps: a) reacting dimedone with isovaleric acid under the action of a condensing agent to generate an intermediate compound Ivdde-OH as shown in formula (3); b) reacting the intermediate compound Ivdde-OH as shown in formula (3) with N-alpha-tert-butoxycarbonyl-L-lysine under the action of an organic base to obtain an intermediate Boc-Lys(Ivdde)-OH as shown in formula (5); c) reacting the intermediate Boc-Lys(Ivdde)-OH as shown in formula (5) with an acid to obtain an intermediate Lys(Ivdde)-OH as shown in formula (6); d) reacting the intermediate Lys(Ivdde)-OH as shown in formula (6) with fluorenylmethoxycarbonyl succinimide under the action of an inorganic base to obtain Fmoc-Lys(Ivdde)-OH as shown in formula (8). The method provided by the present application uses dimedone as the starting material, and synthesizes Fmoc-Lys(Ivdde)-OH through a two-step condensation reaction, a Boc removal reaction, and an Fmoc reaction, a total of 4 steps, which simplifies the process, is simple to operate, and has mild reaction conditions, reduces the difficulty of industrial production, does not involve precious raw materials, and is conducive to controlling production costs. The obtained Fmoc-Lys(Ivdde)-OH has high purity and high yield. The experimental results show that the purity of Fmoc-Lys(Ivdde)-OH prepared by the method provided by the present application is as high as 99% or more, and the yield is as high as 75% or more (calculated as Boc-Lys). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1HPLC spectrum of the intermediate Boc-Lys(Ivdde)-OH prepared in Example 2 of the present application;

[0034] Figure 2 HPLC spectrum of Fmoc-Lys(Ivdde)-OH prepared in Example 2 of the present application;

[0035] Figure 3 This is the infrared spectrum of Fmoc-Lys(Ivdde)-OH prepared in Example 2 of the present application;

[0036] Figure 4 This is the NMR spectrum of Fmoc-Lys(Ivdde)-OH prepared in Example 2 of the present application. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0039] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "multiple" means two or more, unless otherwise clearly and specifically defined. In addition, the terms "installed", "connected" and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal connection of two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0040] The present application provides a method for preparing Fmoc-Lys(Ivdde)-OH, comprising the following steps:

[0041] a) dimedone and isovaleric acid react under the action of a condensing agent to generate an intermediate compound Ivdde-OH shown in formula (3);

[0042]

[0043] b) the intermediate compound Ivdde-OH represented by formula (3) reacts with N-alpha-tert-butoxycarbonyl-L-lysine under the action of an organic base to obtain the intermediate Boc-Lys(Ivdde)-OH represented by formula (5);

[0044]

[0045] c) reacting the intermediate Boc-Lys(Ivdde)-OH represented by formula (5) with an acid to obtain the intermediate Lys(Ivdde)-OH represented by formula (6);

[0046]

[0047] d) reacting the intermediate Lys(Ivdde)-OH represented by formula (6) with fluorenylmethoxycarbonyl succinimide under the action of an inorganic base to obtain Fmoc-Lys(Ivdde)-OH represented by formula (8);

[0048]

[0049] The present invention uses dimedone as a starting material, and synthesizes Fmoc-Lys(Ivdde)-OH through a two-step condensation reaction, a Boc removal reaction, and an Fmoc reaction, a total of four steps of reactions, which simplifies the process, is simple to operate, and has 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-Lys(Ivdde)-OH has high purity and high yield.

[0050] In the present application, dimedone and isovaleric acid are used as raw materials, and react under the action of a condensing agent to generate an intermediate compound Ivdde-OH shown in formula (3), and the reaction process is as follows:

[0051]

[0052] Dimedone has the structure of formula (1), and the present application has no special restrictions on its source; isovaleric acid has the structure of formula (2), and the present application has no special restrictions on its source.

[0053] In some specific implementations, in step a), the condensing agent includes but is not limited to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) or dicyclohexylcarbodiimide (DCC), etc., and can be one or more thereof. When the condensing agent is a combination of multiple substances, the present application has no special restrictions on its proportion. In some specific implementations, the condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) or dicyclohexylcarbodiimide (DCC).

[0054] In some specific implementations, in step a), the reaction medium is an organic solvent. In some specific implementations, the organic solvent is selected from ethyl acetate, acetone or tetrahydrofuran.

[0055] In some specific implementations, the molar ratio of dimedone, isovaleric acid and condensing agent is 1:(0.8-1.3):(1-1.5), preferably 1:1:1.1.

[0056] Specifically, the present application first mixes dimedone, isovaleric acid and an organic solvent uniformly, then adds a condensing agent to react, and obtains the intermediate compound Ivdde-OH shown in formula (3). In some specific implementations, the condensing agent is added at 15°C to 20°C for reaction. In some specific implementations, the reaction temperature is room temperature, for example, 25°C to 35°C, preferably 28°C to 32°C, and the reaction time is 12h to 16h, preferably 13h to 15h.

[0057] After the reaction is completed, the obtained reaction product is subjected to post-treatment, specifically, comprising the following steps:

[0058] Add water to the reaction system, let stand and separate the layers, and collect the organic layer;

[0059] The organic layer was dried and concentrated to obtain the intermediate compound Ivdde-OH represented by formula (3).

[0060] The present application has no particular limitation on the drying method, for example, sodium sulfate can be used for drying. After drying and concentration, the intermediate compound Ivdde-OH shown in formula (3) can be obtained, which can be directly used in the next step without further purification.

[0061] After obtaining the intermediate compound Ivdde-OH shown in formula (3), it is reacted with N-alpha-tert-butoxycarbonyl-L-lysine under the action of an organic base to obtain the intermediate Boc-Lys(Ivdde)-OH shown in formula (5). The reaction process is as follows:

[0062]

[0063] N-alpha-tert-butoxycarbonyl-L-lysine (Boc-Lys) has the structure of formula (4). The present application has no special restrictions on its source and it can be purchased from the market.

[0064] In some specific implementations, the organic base includes but is not limited to triethylamine or N, N-diisopropylethylamine, and can be one or more thereof. When the organic base is multiple thereof, the present application has no particular restrictions on the ratio of each substance. In some specific implementations, the organic base is triethylamine or N, N-diisopropylethylamine.

[0065] 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, methanol or ethanol.

[0066] In some specific implementations, the molar ratio of Boc-Lys, Ivdde-OH, and the organic base is 1:(1-1.5):(1.1-2.0), preferably 1:1.1:1.5.

[0067] Specifically, the present application mixes Boc-Lys, Ivdde-OH and an organic base with an organic solvent, and heats the mixture under reflux to obtain the intermediate Boc-Lys(Ivdde)-OH shown in formula (5). In some specific implementations, the reaction temperature is 65° C. to 75° C., and the reaction time is 12 h to 14 h. In some specific implementations, the reaction is carried out under stirring.

[0068] After the reaction is completed, the obtained reaction product is subjected to post-treatment, specifically, comprising the following steps:

[0069] The reaction system is concentrated to dryness, and after cooling, water, an organic solvent and an acid are added, and the organic layer is collected after standing and separating the liquids;

[0070] The organic layer is washed, dried, concentrated, crystallized, filtered, and dried again to obtain the intermediate Boc-Lys(Ivdde)-OH represented by formula (5).

[0071] The present application first concentrates the reaction system to dryness, preferably cools it to room temperature and then adds water, an organic solvent and an acid; wherein the organic solvent may be ethyl acetate, etc., and the acid is added to adjust the pH value of the system to 4-5. The present application has no special restrictions on the type of the acid, which may be citric acid. After standing and separating the liquids, the present application has no restrictions on the washing method of the collected organic layer, for example, it can be washed 3 times with saturated sodium chloride. The present application has no special restrictions on the drying method, for example, it can be dried with sodium sulfate. After drying, filter, concentrate the obtained filtrate until a large amount of solid precipitates, cool it again and crystallize it. The present application preferably adds a solvent such as petroleum ether for crystallization, crystallizes for 1h to 3h, filters the obtained product, and dries it again to obtain the intermediate Boc-Lys(Ivdde)-OH shown in formula (5). The present application has no special restrictions on the drying here, and it can be dried at 50℃ to 55℃.

[0072] After obtaining the intermediate Boc-Lys(Ivdde)-OH shown in formula (5), it is reacted with an acid to obtain the intermediate Lys(Ivdde)-OH shown in formula (6). The reaction process is as follows:

[0073]

[0074] In some specific implementations, the acid is selected from hydrochloric acid, which functions to remove the Boc protecting group.

[0075] In some specific implementations, the molar ratio of Boc-Lys(Ivdde)-OH to acid is 1:(5-8), preferably 1:7.

[0076] In some specific implementations, the reaction medium is an organic solvent, including but not limited to tetrahydrofuran and the like.

[0077] Specifically, the present application mixes an organic solvent and an acid, and then mixes with the intermediate Boc-Lys(Ivdde)-OH shown in formula (5) and reacts to obtain the intermediate Lys(Ivdde)-OH shown in formula (6). In some specific implementations, the reaction temperature is room temperature, such as 20°C to 25°C, and the reaction time is 6h to 8h.

[0078] After the reaction is completed, a base is added to the obtained reaction system to adjust the pH value to 6-7. After standing and stratification, the aqueous layer is collected to obtain the intermediate Lys(Ivdde)-OH represented by formula (6), which can be directly subjected to the next step of reaction without further treatment.

[0079] After obtaining the intermediate Lys(Ivdde)-OH shown in formula (6), it is reacted with fluorenylmethoxycarbonyl succinimide under the action of an inorganic base to obtain Fmoc-Lys(Ivdde)-OH shown in formula (8). The reaction process is as follows:

[0080]

[0081] Fluorenylmethoxycarbonyl succinimide (Fmoc-Osu) has the structure of formula (7), and the present application has no particular limitation on its source.

[0082] In some specific implementations, the inorganic base includes but is not limited to sodium carbonate, sodium bicarbonate, potassium carbonate, etc., and can be one or more of them. When the inorganic base is more than one of them, the present application has no special restrictions on the ratio of each substance.

[0083] In some specific implementations, the reaction is carried out in an organic solvent, including but not limited to ethyl acetate, acetone or tetrahydrofuran.

[0084] In some specific implementations, the molar ratio of Lys(Ivdde)-OH, Fmoc-Osu, and inorganic base is 1:(0.7-1.0):(1.2-1.5), preferably 1:0.8:1.4.

[0085] Specifically, the present application mixes Lys(Ivdde)-OH aqueous solution, organic solvent and inorganic base uniformly and then adds Fmoc-Osu to react to obtain Fmoc-Lys(Ivdde)-OH shown in formula (8). In some specific implementations, the temperature for adding Fmoc-Osu is preferably 25°C to 30°C, and the temperature for the reaction is preferably 25°C to 30°C. 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. In some specific implementations, the reaction time is 3h to 4h. In some specific implementations, the reaction is carried out under stirring.

[0086] After the reaction is completed, post-treatment is preferably performed, which specifically comprises the following steps:

[0087] Acid was added to the reaction system to adjust the pH to 2-3, and the organic layer was collected after separation;

[0088] The organic layer is washed, dried, concentrated, crystallized, centrifuged and dried to obtain Fmoc-Lys(Ivdde)-OH represented by formula (8).

[0089] The present application has no special restrictions on the acid, which can be concentrated 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 and an organic solvent can be added for crystallization, and the organic solvent includes but is not limited to petroleum ether, and the crystallization time is preferably 1h to 3h. The present application has no special restrictions on the drying after centrifugation, for example, it can be dried at 50°C to 55°C.

[0090] 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-Lys(Ivdde)-OH, with a purity of more than 99% and a yield of more than 75% (calculated as Boc-Lys).

[0091] The preparation method of Fmoc-Lys(Ivdde)-OH provided in the present application is described in detail below with reference to the examples.

[0092] Example 1

[0093] (1) Preparation of Ivdde-OH:

[0094] 650 g of dichloromethane was added to a 1L three-necked flask, and 100 g of dimedone represented by formula (1) and 73 g of isovaleric acid represented by formula (2) were added under stirring, and the mixture was completely dissolved. 150 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl) was added, and the temperature was controlled at 15°C to 20°C. After the addition was completed, the temperature was controlled at 25°C to 30°C for 13 hours. The TLC raw material dimedone was completely reacted. 200 g of water was added to the system and stirred to dissolve clearly. The mixture was allowed to stand for stratification, and the dichloromethane layer was collected. Sodium sulfate was added to the dichloromethane layer, dried, and filtered. The filtrate was concentrated to dryness to obtain 121 g of an oily substance, which was Ivdde-OH represented by formula (3). The yield was 81.2%, and the oily substance was directly used in the next step without detection and purification. The reaction process was as follows:

[0095]

[0096] (2) Preparation of Boc-Lys(Ivdde)-OH:

[0097] 726 g of methanol was added to a 2 L three-necked flask, and 121 g of N-alpha-tert-butyloxycarbonyl-L-lysine (Boc-Lys) represented by formula (4), 121 g of Ivdde-OH prepared in step (1), and 95 g of N, N-diisopropylethylamine, heated to an internal temperature of 70℃~75℃ and reacted for 13h, TLC raw material Boc-Lys was completely reacted; the methanol in the reaction system was concentrated to dryness, cooled to 20℃~30℃, 200g ethyl acetate and 200g water were added to the reaction solution, and the system pH was acidified to 4~5 with citric acid, and then the solution 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, and after washing, sodium sulfate was added and dried, and then filtered and concentrated until a large amount of solid precipitated, cooled to 20℃~25℃, 200g petroleum ether was added and crystallized for 2h, filtered, and dried at 50℃~55℃ to obtain 198g of intermediate Boc-Lys(Ivdde)-OH solid, with a yield of 89% and a HPLC purity of 99.18%. The reaction process is as follows:

[0098]

[0099] (3) Preparation of Lys(Ivdde)-OH:

[0100] 560 g of tetrahydrofuran and 612 g of 6N hydrochloric acid were added to a 2L three-necked flask, and 198 g of Boc-Lys(Ivdde)-OH prepared in step (2) was added under stirring. The temperature was controlled to 20°C to 25°C for reaction for 6 h. The raw material Boc-Lys(Ivdde)-OH was completely reacted as controlled by TLC. Sodium carbonate was slowly added to the system to adjust the pH to 6 to 7. After the pH was adjusted, the system was allowed to stand for stratification. The water layer, i.e., the aqueous solution of Lys(Ivdde)-OH shown in formula (6), was collected. The next step of reaction was directly carried out without taking it out. The reaction process was as follows:

[0101]

[0102] (4) Preparation of Fmoc-Lys(Ivdde)-OH:

[0103] Add the Lys(Ivdde)-OH aqueous solution prepared in step (3) into a 2L three-necked flask, add 750g of ethyl acetate and 118g of sodium carbonate, stir evenly, control the temperature at 25°C to 30°C, add 130g of fluorenylmethoxycarbonyl succinimide (Fmoc-Osu), react for 3h, and TLC shows that Fmoc-Osu is completely reacted; adjust the pH of the system to 2-3 with concentrated hydrochloric acid, then stand and separate the liquids, collect the ethyl acetate layer, wash the ethyl acetate layer with saturated sodium chloride for 3 times, add sodium sulfate after washing, dry, filter and concentrate until a large amount of solid precipitates, cool to 20°C to 25°C, add 350g of petroleum ether for crystallization for 2h, filter, and dry at 50°C to 55°C to obtain 182g of intermediate Fmoc-Lys(Ivdde)-OH solid, with a yield of 82.18% and a purity of 99.69% by HPLC. The reaction process is as follows:

[0104]

[0105] Example 2

[0106] (1) Preparation of Ivdde-OH:

[0107] 150 kg of dichloromethane was added to a 500 L reactor, and 23 kg of dimedone and 16.8 kg of isovaleric acid were added under stirring and completely dissolved. 37.1 kg of dicyclohexylcarbodiimide (DCC) was added, and the temperature was controlled at 15°C to 20°C. After the addition was completed, the temperature was controlled at 25°C to 30°C for 14 hours. The TLC raw material dimedone was completely reacted; the insoluble matter (DCU) in the system was filtered, and the filtrate was collected. The filtrate was directly concentrated to dryness to obtain 31 kg of oily substance with a yield of 84.23%, which was directly used in the next step without detection and purification.

[0108] (2) Preparation of Boc-Lys(Ivdde)-OH:

[0109] 186 kg of ethanol was added to a clean, anhydrous 500 L reactor, and 31 kg of Boc-Lys, 31 kg of Ivdde-OH prepared in step (1), and 19 kg of triethylamine were added under stirring. The temperature was raised to 70°C to 75°C and the reaction was continued for 13 hours. The raw material Boc-Lys was completely reacted by TLC. The ethanol in the reaction system was concentrated to dryness, and the temperature was lowered 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. The reaction solution 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°C to 25°C, 100 kg of petroleum ether was added, and crystallized for 2 hours. The reaction solution was centrifuged and dried at 50°C to 55°C to obtain 53 kg of solid intermediate Boc-Lys(Ivdde)-OH. Figure 1 , Figure 1 This is the HPLC spectrum of the intermediate Boc-Lys(Ivdde)-OH prepared in Example 2 of the present application. Yield: 93%, HPLC purity: 99.28%;

[0110] (3) Preparation of Lys(Ivdde)-OH:

[0111] To a clean 500L reactor, add 150kg of tetrahydrofuran and 164kg of 6N hydrochloric acid, and add 53kg of Boc-Lys(Ivdde)-OH prepared in step (2) under stirring. Control the temperature to 20°C-25°C and react for 7h. TLC shows that the raw material Boc-Lys(Ivdde)-OH is completely reacted. Slowly add sodium carbonate to the system to adjust the pH to 6-7. After the pH is adjusted, stand and separate the layers. Collect the water layer, i.e., the Lys(Ivdde)-OH aqueous solution. There is no need to take it out and directly proceed to the next step.

[0112] (4) Preparation of Fmoc-Lys(Ivdde)-OH:

[0113] Add the Lys(Ivdde)-OH aqueous solution prepared in step (3) to a 1000L reactor, add 200kg of ethyl acetate, add 31.5kg of sodium carbonate, stir evenly, control the temperature at 25℃~30℃, add 35kg of Fmoc-Osu, react for 3h, and TLC Fmoc-Osu is completely reacted; adjust the pH of the system to 2-3 with concentrated hydrochloric acid, then stand and separate, 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℃~25℃, add 100kg of petroleum ether for crystallization for 2h, centrifuge, and dry at 50℃~55℃ to obtain 50kg of intermediate Fmoc-Lys(Ivdde)-OH solid, yield: 83.9%, HPLC purity: 99.70%. See Figure 2 , Figure 3 and Figure 4 , Figure 2 This is the HPLC spectrum of Fmoc-Lys(Ivdde)-OH prepared in Example 2 of the present application. Figure 3 This is the infrared spectrum of Fmoc-Lys(Ivdde)-OH prepared in Example 2 of the present application, Figure 4 This is the NMR spectrum of Fmoc-Lys(Ivdde)-OH prepared in Example 2 of the present application.

[0114] Example 3

[0115] Referring to the preparation method provided in step (1) of Example 1, only the type of condensing agent was changed, and the other preparation steps and process parameters were the same as those of step (1) of Example 1. The yield and purity of step (1) are shown in Table 1.

[0116] Table 1

[0117] Condensation agent Yield Example 1 EDCl 81.2％ Example 3 DCC 84.23％

[0118] Example 4

[0119] Referring to the preparation method provided in step (2) of Example 1, only the type of organic solvent was changed, and the other preparation steps and process parameters were the same as those of step (2) of Example 1. The yield and purity of step (2) are shown in Table 2.

[0120] Table 2

[0121] Organic solvents Reaction time Yield purity Example 1 Methanol 13h 89％ 99.18％ Example 2 Ethanol 13h 93％ 99.28％ Example 4 Acetonitrile 16h 85％ 98.56％

[0122] Examples 5-6, Comparative Examples 1-2

[0123] Referring to the preparation method provided in step (2) of Example 1, only the reaction temperature was changed, and the other preparation steps and process parameters were the same as those of step (2) of Example 1. The yield and purity of step (2) are shown in Table 3.

[0124] Table 3

[0125] Reaction temperature Reaction time Yield purity Example 1 70℃～75℃ 13h 89％ 99.18％ Example 5 76℃～80℃ 12h 85％ 99.06％ Example 6 65℃～69℃ 16h 85％ 98.98％ Comparative Example 1 50℃～55℃ 34h 63％ 97.46％ Comparative Example 2 30℃～35℃ No complete response within 48 hours 36％ 68.53％

[0126] Embodiment 7-8

[0127] Referring to the preparation method provided in step (4) of Example 1, only the type of inorganic base was changed, and the other preparation steps and process parameters were the same as those of step (4) of Example 1. The yield and purity of step (4) are shown in Table 4 below.

[0128] Table 4

[0129] Organic solvents Reaction time Yield purity Example 1 Sodium carbonate 3h 82.18％ 99.69％ Example 7 Potassium carbonate 2h 80.12％ 99.38％ Example 8 Sodium bicarbonate 18h 78.3％ 99.45％

[0130] Embodiments 9-10

[0131] Referring to the preparation method provided in step (4) of Example 1, only the type of organic solvent was changed, and the other preparation steps and process parameters were the same as those of step (4) of Example 1. The yield and purity of step (4) are shown in Table 5.

[0132] Table 5

[0133] Organic solvents Yield purity Example 1 Ethyl acetate 82.18％ 99.69％ Example 9 acetone 77.23％ 99.58％ Example 10 Tetrahydrofuran 78.68％ 99.63％

[0134] In summary, the present invention provides a method for preparing Fmoc-Lys(Ivdde)-OH, which is suitable for scale-up. The synthesis method requires simple equipment, has good reaction efficiency, low production cost, and the purity of the final product is greater than 99.0%, and the yield can reach more than 75% (calculated as Boc-Lys).

[0135] 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-Lys(Ivdde)-OH, comprising the following steps: a) dimedone and isovaleric acid react under the action of a condensing agent to generate an intermediate compound Ivdde-OH shown in formula (3); b) the intermediate compound Ivdde-OH represented by formula (3) reacts with N-alpha-tert-butoxycarbonyl-L-lysine under the action of an organic base to obtain the intermediate Boc-Lys(Ivdde)-OH represented by formula (5); c) reacting the intermediate Boc-Lys(Ivdde)-OH represented by formula (5) with an acid to obtain the intermediate Lys(Ivdde)-OH represented by formula (6); d) reacting the intermediate Lys(Ivdde)-OH represented by formula (6) with fluorenylmethoxycarbonyl succinimide under the action of an inorganic base to obtain Fmoc-Lys(Ivdde)-OH represented by formula (8); 2. The preparation method according to claim 1, characterized in that: In step a), the condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or dicyclohexylcarbodiimide; The molar ratio of dimedone, isovaleric acid and condensing agent is 1:(0.8-1.3):(1-1.5).

3. The preparation method according to claim 2, characterized in that: In step a), the reaction medium is dichloromethane, acetone or tetrahydrofuran; The reaction temperature is room temperature and the reaction time is 12 h to 16 h.

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

5. The preparation method according to claim 4, characterized in that: In step b), the reaction medium is acetonitrile, methanol or ethanol; The reaction temperature is 65°C to 75°C, and the reaction time is 12h to 14h.

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

7. The preparation method according to claim 6, characterized in that: In step c), the reaction temperature is room temperature and the reaction time is 6 h to 8 h.

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

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

10. The preparation method according to claim 8, characterized in that: In step d), the reaction medium is ethyl acetate, acetone or tetrahydrofuran; The reaction temperature is 25°C to 30°C, and the reaction time is 3h to 4h.

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