Method for preparing fmoc-lys(MTT)-oh

Abstract

Provided in the present application is a method for preparing Fmoc-Lys(Mtt)-OH, which method comprises: in the presence of an organic base, bringing Fmoc-Lys·HCl as represented by formula (1), which is used as a starting material, into reaction with Mtt-Cl as represented by formula (II) to synthesize Fmoc-Lys(Mtt)-OH as represented by formula (III). The method has a simplified process, easy operation and mild reaction conditions, reduces the difficulty of industrial production, does not involve expensive raw materials, and helps in controlling production costs. The obtained Fmoc-Lys(Mtt)-OH exhibits a high purity and a high yield. Experimental results show that Fmoc-Lys(Mtt)-OH prepared by means of the preparation method provided in the present application has a yield of 80% or more and a purity of 99% or more.

Description

A method for preparing Fmoc-Lys(Mtt)-OH

[0001] This application claims priority to Chinese Patent Application No. 202411626542.X, filed on November 14, 2024, entitled “A method for preparing Fmoc-Lys(Mtt)-OH”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of drug synthesis technology, specifically relating to a method for preparing Fmoc-Lys(Mtt)-OH. Background Technology

[0003] Fmoc-Lys(Mtt)-OH is a commonly used intermediate in the synthesis of peptides such as smegglutinin. Its Chinese name is fluorenylmethoxycarbonyl-N'-4-methyltriphenyl-lysine, and its molecular formula is C2. 41 H 40 N₂O₄, CAS number 167393-62-6, has the following structural formula:

[0004] In existing technologies, the common method for preparing Fmoc-Lys(Mtt)-OH is as follows: Lys and Mtt-Cl are first reacted to synthesize Mtt-Lys(Mtt)-OH. One of the Mtt atoms is then removed with acid to obtain Lys(Mtt)-OH. Finally, Fmoc-osu protection is applied to obtain Fmoc-Lys(Mtt)-OH. However, this method has a low yield, requires a large amount of Mtt-Cl, and incurs high raw material costs, making it unsuitable for modern industrial production. The specific reaction is as follows: Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for preparing Fmoc-Lys(Mtt)-OH. The preparation method provided in this application is simple, the reaction conditions are mild, and the yield and purity of the obtained product are both high.

[0006] This application provides a method for preparing Fmoc-Lys(Mtt)-OH, comprising the following steps:

[0007] The compound shown in formula (1) reacts with the compound shown in formula (II) under the action of an organic base to give Fmoc-Lys(Mtt)-OH shown in formula (III);

[0008] In some specific implementations, the organic base is selected from at least one of triethylamine or N,N-diisopropylethylamine.

[0009] In some specific implementations, the molar ratio of the compound shown in formula (1), the compound shown in formula (II), and the organic base is 1:(0.8 to 1.3):(2 to 2.5).

[0010] In some specific implementations, the organic solvent for the reaction is selected from dichloromethane, methanol, ethanol, or tetrahydrofuran.

[0011] In some specific implementations, the reaction temperature is 5℃~25℃ and the time is 2h~10h.

[0012] In some specific implementations, the compound shown in formula (1) is mixed evenly with an organic solvent, then the compound shown in formula (II) is added and mixed evenly, and then an organic base is added and reacted.

[0013] In some specific implementations, the temperature at which the compound shown in formula (II) is added is 5°C to 25°C.

[0014] In some specific implementations, the organic base is added dropwise.

[0015] Some specific implementations also include post-processing of the obtained reaction products.

[0016] In some specific implementations, the post-processing includes:

[0017] The reaction product was washed successively with citric acid solution and brine, dried, filtered and crystallized to obtain Fmoc-Lys(Mtt)-OH as shown in formula (III).

[0018] This application uses Fmoc-Lys·HCl (as shown in formula (I)) as the starting material and reacts it with Mtt-Cl (as shown in formula (II)) in the presence of an organic base to synthesize Fmoc-Lys(Mtt)-OH (as shown in formula II (I)). This simplifies the process, is easy to operate, and uses mild reaction conditions, reducing the difficulty of industrial production. It does not involve expensive raw materials, which helps control production costs. The obtained Fmoc-Lys(Mtt)-OH has high purity and high yield. Experimental results show that the preparation method provided in this application yields Fmoc-Lys(Mtt)-OH with a purity of over 80% and over 99%. Attached Figure Description

[0019] Figure 1 shows the high performance liquid chromatography (HPLC) spectrum of the product prepared in Example 1;

[0020] Figure 2 shows the infrared spectrum of the product prepared in Example 1;

[0021] Figure 3 shows the high performance liquid chromatography (HPLC) chromatogram of the product prepared in Example 2;

[0022] Figure 4 shows the infrared spectrum of the product prepared in Example 2;

[0023] Figure 5 shows the high performance liquid chromatography (HPLC) chromatogram of the product prepared in Example 3;

[0024] Figure 6 shows the infrared spectrum of the product prepared in Example 3;

[0025] Figure 7 shows the NMR spectrum of the product prepared in Example 3. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," and "counterclockwise," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention.

[0028] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the stated features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified. Furthermore, the terms "installed," "connected," and "linked" should be interpreted broadly; for example, they may refer to a fixed connection, a detachable connection, or an integral connection; they may refer to a mechanical connection or an electrical connection; they may refer to a direct connection or an indirect connection through an intermediate medium; and they may refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0029] This application provides a method for preparing Fmoc-Lys(Mtt)-OH, comprising the following steps:

[0030] The compound shown in formula (1) reacts with the compound shown in formula (II) under the action of an organic base to give Fmoc-Lys(Mtt)-OH shown in formula (III);

[0031] This application uses the compound shown in formula (1) and the compound shown in formula (II) as raw materials, wherein the compound shown in formula (1) is Fmoc-Lys·HCl and the compound shown in formula (II) is Mtt-Cl. This application does not have any special restrictions on their source and they can be purchased on the market.

[0032] In one specific implementation, this application first mixes the compound shown in formula (1) with an organic solvent until homogeneous, then adds the compound shown in formula (II) and mixes until homogeneous, then adds an organic base and reacts to obtain Fmoc-Lys(Mtt)-OH shown in formula (III).

[0033] This application first mixes the compound represented by formula (I) with an organic solvent under stirring conditions. This application does not impose any special limitations on the stirring parameters; as long as the compound of formula (I) and the organic solvent are mixed evenly, it is acceptable. In some specific implementations, the organic solvent for the reaction is selected from dichloromethane, methanol, ethanol, or tetrahydrofuran. This application does not impose any special limitations on the amount of the organic solvent used; any amount that facilitates the reaction is acceptable.

[0034] The compound shown in formula (I) is mixed with an organic solvent, and then the compound shown in formula (II) is added under stirring. In some specific implementations, the compound shown in formula (II) is added at the reaction temperature, for example, 5°C to 25°C.

[0035] After the compound shown in formula (II) is mixed thoroughly, an organic base is added to initiate the reaction, preferably by dropwise addition. In some specific implementations, the organic base is selected from at least one of triethylamine or N,N-diisopropylethylamine, preferably N,N-diisopropylethylamine. In some specific implementations, the reaction temperature is 5°C to 25°C, preferably 15°C to 25°C, and the reaction time is 2h to 10h, preferably 2h to 6h. In this application, a reaction temperature below 5°C results in incomplete reaction, while a reaction temperature above 25°C increases impurities, leading to a lower yield.

[0036] In some specific implementations, the molar ratio of the compound shown in formula (1), the compound shown in formula (II), and the organic base is 1:(0.8 to 1.3):(2 to 2.5), preferably 1:1.05:2.5.

[0037] After the reaction is complete, the obtained reaction product undergoes post-processing. This application does not specifically limit the method of post-processing, which may include the following steps:

[0038] The reaction product was washed successively with citric acid solution and brine, dried, filtered and crystallized to obtain Fmoc-Lys(Mtt)-OH as shown in formula (III).

[0039] After obtaining the reaction product, it is first washed with an aqueous citric acid solution, the mass concentration of which is 5%–15%, preferably 10%, and the washing is preferably performed twice. After washing with the citric acid solution, it is washed again with saturated brine, and the washing is preferably performed three times. After washing, the obtained organic phase is dried, for example, using anhydrous sodium sulfate, filtered, and then the system is concentrated.

[0040] The concentrated reaction product is then crystallized, with the crystallization solvent including, but not limited to, petroleum ether. Specifically, the concentrated reaction product is cooled to 20°C–25°C, then a solvent is added, and the temperature is lowered to 0°C–5°C to induce crystallization. After filtration and drying, the final product is obtained. In some specific implementations, drying is preferably performed at 50°C–55°C.

[0041] This application uses Fmoc-Lys·HCl (as shown in formula (I)) as the starting material and reacts it with Mtt-Cl (as shown in formula (II)) in the presence of an organic base to synthesize Fmoc-Lys(Mtt)-OH (as shown in formula II (I)). This simplifies the process, is easy to operate, and uses mild reaction conditions, reducing the difficulty of industrial production. It does not involve expensive raw materials, which helps control production costs. The obtained Fmoc-Lys(Mtt)-OH has high purity and high yield. Experimental results show that the preparation method provided in this application yields Fmoc-Lys(Mtt)-OH with a yield of over 80% and a purity of over 99%.

[0042] The preparation method of Fmoc-Lys(Mtt)-OH provided in this application will be described in detail below with reference to the embodiments.

[0043] Example 1

[0044] Add 405g of dichloromethane to a 2L three-necked flask, then add 40.5g of Fmoc-Lys·HCl with stirring. Maintain the temperature at 15℃~25℃, add 30.8g of Mtt-Cl, and then add 27.1g of N,N-diisopropylethylamine dropwise. After the addition is complete, maintain the temperature at 15℃~25℃ and react for 4 hours. The reaction was completed by TLC with Fmoc-Lys·HCl. Wash the reaction solution twice with 10% citric acid aqueous solution (80g / time), and wash the organic phase three times with saturated brine (80g / time). Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate to a final volume of 130g. Cool to 20℃~25℃, add 100g of petroleum ether, and a large amount of solid precipitates. Cool to 0~5℃ to crystallize for 2h~4h, filter, and dry at 50℃~55℃ to obtain 52.1g of white solid, yield 83.4%, purity 99.77%.

[0045] The white solid was analyzed, and the results are shown in Figures 1 and 2. Figure 1 is the high-performance liquid chromatography (HPLC) chromatogram of the product prepared in Example 1, and Figure 2 is the infrared spectrum of the product prepared in Example 1. As can be seen from Figures 1 and 2, the white solid prepared in this application is Fmoc-Lys(Mtt)-OH.

[0046] Example 2

[0047] Add 405g of methanol to a 2L three-necked flask, then add 40.5g of Fmoc-Lys·HCl while stirring. Maintain the temperature at 15℃~25℃, then add 30.8g of Mtt-Cl, followed by dropwise addition of 27.1g of N,N-diisopropylethylamine. After the addition is complete, maintain the temperature at 15℃~25℃ and react for 5 hours. TLC is then used for the reaction. After the Fmoc-Lys·HCl reaction was complete, 80g of water was added to the reaction solution, and the pH was adjusted to 7-7.5 with citric acid. The methanol in the system was concentrated to dryness, and 240g of dichloromethane was added. The reaction solution was washed twice (80g / time) with 10% citric acid aqueous solution, and the organic phase was washed three times (80g / time) with saturated brine. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to a remaining 133g. The temperature was lowered to 20℃-25℃, and 100g of petroleum ether was added, resulting in the precipitation of a large amount of solid. The temperature was lowered to 0℃-5℃ for 2-4 hours to crystallize, filtered, and dried at 50℃-55℃ to obtain 53.3g of white solid, with a yield of 85.3% and a purity of 99.82%.

[0048] The white solid was analyzed, and the results are shown in Figures 3 and 4. Figure 3 is the high-performance liquid chromatography (HPLC) spectrum of the product prepared in Example 2, and Figure 4 is the infrared spectrum of the product prepared in Example 2. As can be seen from Figures 3 and 4, the white solid prepared in this application is Fmoc-Lys(Mtt)-OH.

[0049] Example 3

[0050] Add 1500 kg of dichloromethane to a 3000 L enamel-lined reactor, then add 150 kg of Fmoc-Lys·HCl while stirring. Maintain the temperature at 15℃~25℃, then add 114 kg of Mtt-Cl. Next, add 119.5 kg of N,N-diisopropylethylamine dropwise. After the addition is complete, maintain the temperature at 15℃~25℃ and react for 6 hours. TLC is then used for analysis. The reaction of Fmoc-Lys·HCl was completed. The reaction solution was washed twice (300 kg / time) with 10% citric acid aqueous solution, and the organic phase was washed three times (300 kg / time) with saturated brine. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to a final volume of 300–400 L. The solution was cooled to 20–25 °C, and 300 kg of petroleum ether was added, resulting in the precipitation of a large amount of solid. The solution was then cooled to 0–5 °C for 2–4 hours to allow crystallization. After filtration, the solution was dried at 50–55 °C to obtain 191.65 kg of white solid, with a yield of 82.8% and a purity of 99.81%.

[0051] The white solid was analyzed, and the results are shown in Figures 5, 6, and 7. Figure 5 is the high-performance liquid chromatography (HPLC) spectrum of the product prepared in Example 3, Figure 6 is the infrared spectrum of the product prepared in Example 3, and Figure 7 is the nuclear magnetic resonance (NMR) spectrum of the product prepared in Example 3. As can be seen from Figures 5, 6, and 7, the white solid prepared in this application is Fmoc-Lys(Mtt)-OH.

[0052] Examples 4-5

[0053] This embodiment investigated the effect of organic solvents on the product. Specifically, the preparation method provided in Example 1 was followed, except that the type of organic solvent was changed; all other preparation steps and process parameters remained the same as in Example 1. The product yield is shown in Table 1 below.

[0054] Table 1. Yields and purity of the products prepared in Examples 1-4 of this application.

[0055] Comparative Example 1

[0056] This comparative study investigated the effect of organic bases on the product. Specifically, the preparation method provided in Example 1 was followed, except that the type of organic base was changed; all other preparation steps and process parameters remained the same as in Example 1. The product yields are shown in Table 2 below.

[0057] Table 2. Yields and purity of products prepared in the embodiments and comparative examples of this application.

[0058] Example 6, Comparative Examples 2-4

[0059] This embodiment and comparative example investigated the effect of reaction temperature on the product. Specifically, referring to the preparation method provided in Example 1, only the reaction temperature was changed; all other preparation steps and process parameters were the same as in Example 1. The product yield and reaction time are shown in Table 3 below.

[0060] Table 3. Yields and purity of the products prepared in Examples 6-7 and Comparative Examples 2-3 of this application.

[0061] In summary, this invention provides a method for preparing Fmoc-Lys(Mtt)-OH, which is suitable for scale-up. The synthesis method requires simple equipment, has good reaction efficiency, low production cost, and the final product has a purity >99.0% and a yield of over 80%.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing Fmoc-Lys(Mtt)-OH, comprising the following steps: The compound represented by formula (1) is reacted with the compound represented by formula (II) in the presence of an organic base to obtain the Fmoc-Lys(Mtt)-OH represented by formula (III); 2. The production method according to claim 1, characterized by, The organic base is selected from at least one of triethylamine or N,N-diisopropylethylamine.

3. The production method according to claim 1, characterized by, The molar ratio of the compound shown in formula (1), the compound shown in formula (II), and the organic base is 1:(0.8 to 1.3):(2 to 2.5).

4. The preparation method according to claim 1, characterized in that, The organic solvent used in the reaction is selected from dichloromethane, methanol, ethanol, or tetrahydrofuran.

5. The preparation method according to claim 1, characterized in that, The reaction is carried out at a temperature of 5℃ to 25℃ for a time of 2h to 10h.

6. The method of any one of claims 1 to 5, wherein the method further comprises the step of: The compound shown in formula (1) is mixed evenly with an organic solvent, and then the compound shown in formula (II) is added and mixed evenly. Finally, an organic base is added and the mixture is reacted.

7. The production method according to claim 6, wherein The temperature at which the compound shown in formula (II) is added is 5°C to 25°C.

8. The preparation method according to claim 6, characterized in that, The organic base is added dropwise.

9. The production method according to claim 8, characterized by, It also includes post-processing of the obtained reaction products.

10. The method of claim 9, wherein the method further comprises, The post-processing includes: The reaction product was washed successively with citric acid solution and brine, dried, filtered and crystallized to obtain Fmoc-Lys(Mtt)-OH as shown in formula (III).

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