A method for preparing Fmoc-Lys(iPr,Boc)-OH

Abstract

This invention relates to a method for preparing Fmoc-Lys(iPr,Boc)-OH, belonging to the field of synthetic technology. The preparation method of this invention includes the following steps: S1, Cbz-Lys-OH and acetone undergo a first-stage reaction under the action of an acidic catalyst; then, a second-stage reaction is carried out under the action of a reducing agent; finally, the pH is adjusted to 9-11, and Boc2O is added for a third-stage reaction, followed by salt purification to obtain Cbz-Lys(iPr,Boc)-OH; S2, under H2 conditions, Cbz-Lys(iPr,Boc)-OH undergoes a hydrogenation reaction in an alcohol solvent under the action of a metal catalyst; then, the pH is adjusted to 8-10, and Fmoc-OSu is added for Fmoc protection reaction, followed by salt purification and crystallization purification to obtain Fmoc-Lys(iPr,Boc)-OH. The purity of the product reaches over 99%, and the yield reaches over 75%. The purification process uses salt formation and crystallization operations without column purification, making it suitable for industrial-scale production.

Description

Technical Field

[0001] This invention belongs to the field of synthetic technology, and particularly relates to a method for preparing Fmoc-Lys(iPr,Boc)-OH. Background Technology

[0002] Degarelix is ​​a novel drug developed by Ferring Pharmaceuticals (A / S) for the treatment of advanced prostate cancer. Approved by the FDA in 2008, it is marketed as Firmagon. It is a linear decapeptide containing seven non-natural amino acids, with the peptide sequence Ac-D-2-Nal-D-Phe(4-Cl)-D-3-Pal-Ser-4-Aph(L-Hor)-4-D-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2. Fmoc-Lys(iPr,Boc)-OH is one of the starting materials for the synthesis of degarelix. Patent US2020 / 268729 discloses the synthesis of this compound.

[0003]

[0004] Starting with Fmoc-Lys(Boc)-OH, under trifluoroacetic acid conditions, the Boc group is first removed to obtain Fmoc-Lys-OH. The amino group in this compound condenses with acetone under trifluoroacetic acid conditions to form an imine. NaBH(OAc)3 reduces the imine to Fmoc-Lys(iPr)-OH, and finally, a Boc group is added to obtain Fmoc-Lys(iPr,Boc)-OH. In this synthetic route, during the addition of the Boc group to Fmoc-Lys(iPr)-OH, due to the alkaline conditions, the Fmoc group is removed, producing impurities such as Boc-Lys(iPr,Boc)-OH and Boc-Lys(iPr,Fmoc)-OH. These impurities are similar in properties to the product and are difficult to remove, affecting product purity. The reported overall yield is also low, only 61%. This route uses column chromatography for purification, which is not suitable for industrial production.

[0005] Therefore, it is necessary to develop new methods for preparing Fmoc-Lys(iPr,Boc)-OH to achieve higher yields and purity, and to make them more suitable for industrial production. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for preparing Fmoc-Lys(iPr,Boc)-OH. Using Cbz-Lys-OH as the starting material, the product is obtained through a five-step reaction. The first three steps and the last two steps are each performed in a one-pot cooking process. The intermediate Cbz-Lys(iPr,Boc)-OH is purified by salt formation. The product Fmoc-Lys(iPr,Boc)-OH is purified by salt formation and crystallization, resulting in a product purity exceeding 99% and a yield exceeding 75%. The process is simple to operate, does not require column chromatography purification, and is suitable for industrial-scale production.

[0007] The purpose of this invention is to provide a method for preparing Fmoc-Lys(iPr,Boc)-OH, comprising the following steps:

[0008] S1, Cbz-Lys-OH and acetone undergo a first-stage reaction under the action of an acidic catalyst; then a second-stage reaction is carried out under the action of a reducing agent; finally, the pH is adjusted to 9-11, Boc2O is added to carry out a third-stage reaction, and Cbz-Lys(iPr,Boc)-OH is obtained by salt formation and purification.

[0009] S2. Under H2 conditions, the Cbz-Lys(iPr,Boc)-OH described in S1 undergoes a hydrogenation reaction in an alcohol solvent under the action of a metal catalyst; then the pH is adjusted to 8-10, Fmoc-OSu is added to carry out an Fmoc protection reaction, and the Fmoc-Lys(iPr,Boc)-OH is obtained by salt formation purification and crystallization purification.

[0010] In one embodiment of the present invention, in S1, the reducing agent is selected from one or more of sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, and potassium borohydride.

[0011] In one embodiment of the present invention, in S1, the molar ratio of Cbz-Lys-OH and Boc2O is 1:(1.0-2.0).

[0012] In one embodiment of the present invention, in S1, the reaction time of the first stage is 4h-6h; the reaction time of the second stage is 2h-4h; and the reaction time of the third stage is 3h-5h.

[0013] In one embodiment of the present invention, in S2, the metal catalyst is selected from one or more of Pd / C, Pd(OH)2 / C and Raney nickel.

[0014] In one embodiment of the present invention, in S2, the alcohol solvent is selected from one or more of methanol, ethanol, isopropanol and tert-butanol.

[0015] In one embodiment of the present invention, in S2, the temperature of the hydrogenation reaction is 40°C-60°C, the pressure is 0.2MPa-0.4MPa, and the time is 4h-6h.

[0016] In one embodiment of the present invention, in S2, the duration of the upper Fmoc protection reaction is 2h-4h.

[0017] In one embodiment of the present invention, the molar ratio of Cbz-Lys(iPr,Boc)-OH to Fmoc-OSu is 1:(0.9-1.5).

[0018] In one embodiment of the present invention, the pH adjuster is one or more of potassium carbonate, sodium carbonate, sodium bicarbonate, and potassium bicarbonate.

[0019] In one embodiment of the present invention, the preparation method of Fmoc-Lys(iPr,Boc)-OH includes the following steps:

[0020]

[0021] S1. Cbz-Lys-OH, trifluoroacetic acid, and acetone were stirred at room temperature for 4-6 hours. NaBH4 was added to the reaction solution in portions, and the mixture was stirred for 2-4 hours. The pH was then adjusted to 10 with potassium carbonate aqueous solution. Boc2O was added, and the mixture was stirred for 4 hours. The pH was then adjusted to 3-4 with hydrochloric acid. Most of the acetone was removed by concentration, and the mixture was extracted with an ester solvent, washed with saturated brine, and dicyclohexylamine was added to the filtrate. A solid precipitated out, and the mixture was stirred for 2 hours. The mixture was then filtered, and the filter cake was washed with an ester solvent. The cake was then desalted with an ester solvent and citric acid aqueous solution, and concentrated to obtain Cbz-Lys(iPr,Boc)-OH.

[0022] An alcohol solvent and Pd / C were added to Cbz-Lys(iPr,Boc)-OH, and hydrogenation was carried out in a hydrogenation reactor at 40℃-60℃ for 4-6 hours. Pd / C was removed by filtration, and sodium carbonate aqueous solution was added to adjust the pH to 8-10. Fmoc-OSu was added, and the mixture was stirred at room temperature for 2-4 hours. The pH was adjusted with hydrochloric acid, and most of the alcohol solvent was removed by concentration. Extraction was carried out with an ester solvent. The mixture was washed with saturated brine, and dicyclohexylamine was added to the filtrate, resulting in the precipitation of solids. The mixture was stirred at room temperature for 2 hours, filtered, and the filter cake was washed with an ester solvent. The mixture was desalted with an ester solvent and citric acid aqueous solution. The ester solvent layer was concentrated to remove most of the solvent. The residue was heated to 50℃-60℃, and other organic solvents were added. The mixture was stirred at this temperature until dissolved, and then slowly cooled to 10℃-20℃, resulting in the precipitation of a large amount of solids. The mixture was stirred at this temperature for 2 hours, filtered, washed, and dried to obtain the product Fmoc-Lys(iPr,Boc)-OH.

[0023] Furthermore, the ester solvent is selected from ethyl acetate and / or isopropyl acetate.

[0024] Furthermore, the other organic solvents are selected from one or more of isopropyl ether, methyl ether, n-heptane, and petroleum ether.

[0025] The technical solution of the present invention has the following advantages compared with the prior art:

[0026] (1) The preparation method described in this invention uses Cbz-Lys-OH as raw material and obtains the product by two one-pot cooking processes, passing through the intermediate Cbz-Lys(iPr,Boc)-OH. Compared with the reported routes, since the Cbz protecting group is relatively stable under alkaline and acidic conditions, fewer impurities are generated during the process, and the product can be purified to more than 99% through salt formation and crystallization. Although this route has the addition of Cbz deprotection and Fmoc protection, the yields of Cbz deprotection and Fmoc protection are relatively high, and they are all carried out in a one-pot cooking process together with other reactions. Therefore, the yield is actually higher than that of the reported routes.

[0027] (2) The preparation method described in this invention can prepare the intermediate Cbz-Lys(iPr,Boc)-OH in one pot, which is simple to operate. The impurities are effectively removed by salt purification, which improves the purity of the intermediate and reduces the burden on the purification of subsequent products. The product Fmoc-Lys(iPr,Boc)-OH can also be obtained by one pot. When adding the Fmoc protecting group, there will be some corresponding characteristic impurities, such as Fmoc-β-Ala-OH, HOSu, etc., but they can be removed relatively cleanly and have high purity through salt purification and crystallization purification.

[0028] (3) The preparation method described in this invention adopts two one-pot cooking, which reduces the number of post-processing steps and makes the operation simple. The purity of the obtained Fmoc-Lys(iPr,Boc)-OH reaches more than 99%, and the yield reaches more than 75%. The purification process does not involve column purification, and is suitable for industrial scale-up production. Attached Figure Description

[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0030] Figure 1 The HPLC chromatogram of Fmoc-Lys(iPr,Boc)-OH prepared in Example 1 of this invention;

[0031] Figure 2 This is an HPLC chromatogram of Fmoc-Lys(iPr,Boc)-OH prepared in Comparative Example 1 of this invention. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. It should be understood that the specific embodiments are only used to explain the present invention, but the embodiments are not intended to limit the present invention.

[0033] In this invention, unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] In this invention, unless otherwise stated, the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0035] In this invention, unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0036] In this invention, unless otherwise stated, the meanings of the abbreviations used in the embodiments of this invention are as shown in Table 1:

[0037] Table 1

[0038] abbreviation meaning Cbz-Lys-OH N-Benzyloxycarbonyl-L-L-Lysine <![CDATA[NaBH4]]> Sodium borohydride Fmoc-OSu 9-fluorenemethoxycarbonylsuccinimide <![CDATA[Boc2O]]> Ditert-butyl dicarbonate

[0039] Example 1

[0040] The preparation method of Fmoc-Lys(iPr,Boc)-OH in this embodiment specifically includes the following steps:

[0041] S1. Add Cbz-Lys-OH (140g, 0.5mol), trifluoroacetic acid (74mL, 114g, 1.0mol), and acetone (700mL) to a 2L four-necked reaction flask and stir at room temperature for 4 hours. Add NaBH4 (28g, 0.75mol) in portions to the reaction solution and stir at room temperature for 4 hours. Adjust the pH to 10 with potassium carbonate aqueous solution. Add Boc2O (164g, 0.75mol) and stir the reaction for 4 hours. Use salt... Adjust the pH to 3-4 with acid; concentrate to remove most of the acetone, extract with ethyl acetate (500 mL * 3), combine the organic layers, wash with water (100 mL) and saturated brine (100 mL), add dicyclohexylamine (100 g, 0.55 mol) to the organic layer, a solid precipitates out, stir for 2 h, filter, wash the filter cake with ethyl acetate (100 mL); desalt with ethyl acetate and citric acid aqueous solution, concentrate to obtain the concentrate Cbz-Lys(iPr,Boc)-OH;

[0042] S2. Add the concentrated Cbz-Lys(iPr,Boc)-OH to a 2L hydrogenation reactor, add methanol (1000mL), 10% Pd / C (10g), and hydrogenate at 50℃ and 0.3MPa for 4h. Filter to remove Pd / C, add 10% sodium carbonate aqueous solution to adjust the pH to 8-10 (500mL), add Fmoc-OSu (168g, 0.5mol), and stir at room temperature for 2h. Adjust the pH to 3-4 with hydrochloric acid, concentrate to remove most of the methanol, and extract with ethyl acetate (500mL*3). Combine the organic layers, wash with water (100mL) and saturated brine (100mL). Dicyclohexylamine (82 g, 0.45 mol) was added to the organic layer, resulting in the precipitation of a solid. The mixture was stirred at room temperature for 2 hours, filtered, and the filter cake was washed with ethyl acetate (100 mL). The cake was then desalted with ethyl acetate (500 mL x 3) and an aqueous solution of citric acid. The ethyl acetate layer was concentrated to remove approximately half of the solvent. The residue was heated to 55°C, and isopropyl ether (750 mL) was added. The mixture was stirred at this temperature until dissolved. The temperature was slowly lowered to approximately 15°C, resulting in the precipitation of a large amount of solid. The mixture was stirred at this temperature for 2 hours, filtered, washed with isopropyl ether (100 mL), and dried at approximately 50°C for 8 hours to obtain the product: 193.8 g of white solid, yield 76%, purity 99.5% (see [link to product description]). Figure 1 ).

[0043] Example 2

[0044] The preparation method of Fmoc-Lys(iPr,Boc)-OH in this embodiment specifically includes the following steps:

[0045] S1. Add Cbz-Lys-OH (28g, 0.1mol), trifluoroacetic acid (15mL, 22.8g, 0.2mol), and acetone (150mL) to a 500mL four-necked reaction flask and stir at room temperature for 4h. Add NaBH4 (6g, 0.15mol) in portions to the reaction solution and stir at room temperature for 4h. Then adjust the pH to 10 with potassium carbonate aqueous solution. Add Boc2O (33g, 0.15mol) and stir the reaction for 4h. Adjust the pH to 3-4 with hydrochloric acid; concentrate to remove most of the acetone, extract with ethyl acetate (100 mL * 3), combine the organic layers, wash with water (20 mL) and saturated brine (20 mL), add dicyclohexylamine (20 g, 0.11 mol) to the organic layer, a solid precipitates out, stir for 2 h, filter, wash the filter cake with ethyl acetate (20 mL); desalt with ethyl acetate and citric acid aqueous solution, concentrate to obtain the concentrate Cbz-Lys(iPr,Boc)-OH;

[0046] S2. Add the concentrated Cbz-Lys(iPr,Boc)-OH to a 500mL hydrogenation reactor, along with ethanol (200mL) and 10% Pd / C (2g). Hydrogenate at 50℃ and 0.2MPa for 4h. Filter to remove Pd / C, adjust the pH to 8-10 with 10% sodium carbonate aqueous solution (100mL), and add Fmoc-OSu (33.6g, 0.1mol). Stir at room temperature for 2h. Adjust the pH to 3-4 with hydrochloric acid, concentrate to remove most of the ethanol, and extract with ethyl acetate (100mL*3). Combine the organic layers and wash with water (20mL) and saturated brine (20mL). Dicyclohexylamine (16.4 g, 0.09 mol) was added to the organic layer, and a solid precipitated out. The mixture was stirred at room temperature for 2 h, filtered, and the filter cake was washed with ethyl acetate (100 mL). The cake was then desalted with ethyl acetate (100 mL * 3) and citric acid aqueous solution. The ethyl acetate layer was concentrated to remove about half of the solvent. The residue was heated to 55 °C, and methyl ether (150 mL) was added. The mixture was stirred at this temperature until it dissolved completely. The temperature was then slowly lowered to about 15 °C, and a large amount of solid precipitated out. The mixture was stirred at this temperature for 2 h, filtered, washed with methyl ether (20 mL), and dried at about 50 °C for 8 h to obtain the product, a white solid of 39.7 g, with a yield of 78% and a purity of 99.6%.

[0047] Example 3

[0048] The preparation method of Fmoc-Lys(iPr,Boc)-OH in this embodiment specifically includes the following steps:

[0049] S1. Add Cb2-Lys-OH (28g, 0.1mol), trifluoroacetic acid (15mL, 22.8g, 0.2mol), and acetone (150mL) to a 500mL four-necked reaction flask and stir at room temperature for 4 hours. Add NaBH4 (6g, 0.15mol) to the reaction solution in portions and stir at room temperature for 4 hours. Adjust the pH to 10 with potassium carbonate aqueous solution. Add Boc2O (33g, 0.15mol) and stir the reaction for 4 hours. The pH was adjusted to 3-4 with hydrochloric acid; most of the acetone was removed by concentration, and the mixture was extracted with isopropyl acetate (100 mL * 3). The organic layers were combined and washed with water (20 mL) and saturated brine (20 mL). Dicyclohexylamine (20 g, 0.11 mol) was added to the organic layer, and a solid precipitated out. The mixture was stirred for 2 h, filtered, and the filter cake was washed with isopropyl acetate (20 mL). The mixture was desalted with isopropyl acetate and citric acid aqueous solution and concentrated to obtain the concentrate Cbz-Lys(iPr,Boc)-OH.

[0050] S2. Add the concentrated Cbz-Lys(iPr,Boc)-OH to a 500mL hydrogenation reactor, add methanol (200mL), and 10% Pd / C (2g). Hydrogenate at 50℃ and 0.2MPa for 4h. Filter to remove Pd / C, add 10% sodium carbonate aqueous solution to adjust the pH to 8-10 (100mL), add Fmoc-OSu (33.6g, 0.1mol), and stir at room temperature for 2h. Adjust the pH to 3-4 with hydrochloric acid, concentrate to remove most of the methanol, and extract with isopropyl acetate (100mL*3). Combine the organic layers and wash with water (20mL) and saturated brine (20mL). Dicyclohexylamine (16.4 g, 0.09 mol) was added to the organic layer, and a solid precipitated out. The mixture was stirred at room temperature for 2 h, filtered, and the filter cake was washed with isopropyl acetate (100 mL). It was then desalted with isopropyl acetate (100 mL * 3) and citric acid aqueous solution. The isopropyl acetate layer was concentrated to remove about half of the solvent. The residue was heated to 55 °C, and isopropyl ether (150 mL) was added. The mixture was stirred at this temperature until it dissolved completely. The temperature was then slowly lowered to about 15 °C, and a large amount of solid precipitated out. The mixture was stirred at this temperature for 2 h, filtered, washed with isopropyl ether (20 mL), and dried at about 50 °C for 8 h to obtain the product, a white solid of 40.3 g, with a yield of 79% and a purity of 99.4%.

[0051] Comparative Example 1

[0052] To a 500 mL four-necked reaction flask, add Fmoc-Lys-OH hydrochloride (40.4 g, 0.1 mol), trifluoroacetic acid (15 mL, 22.8 g, 0.2 mol), and acetone (150 mL) and stir at room temperature for 4 h. Add NaBH4 (6 g, 0.15 mol) in portions to the reaction mixture and stir at room temperature for 4 h. Adjust the pH to 9 with potassium carbonate aqueous solution. Add Boc2O (33 g, 0.15 mol) and stir for 4 h. Adjust the pH to 3-4 with hydrochloric acid. Concentrate to remove most of the acetone, extract with ethyl acetate (100 mL * 3), and combine the solutions. The organic layer was washed with water (20 mL) and saturated brine (20 mL). Dicyclohexylamine (20 g, 0.11 mol) was added to the organic layer, resulting in solid precipitation. The mixture was stirred for 2 hours, filtered, and the filter cake was washed with ethyl acetate (20 mL). The mixture was desalted with an aqueous solution of ethyl acetate and citric acid, concentrated to remove approximately half of the ethyl acetate, and then isopropyl ether (150 mL) was added. The mixture was heated until the solution was clear, then slowly cooled to 15°C, resulting in a large amount of solid precipitation. The mixture was stirred at this temperature for 2 hours, filtered, washed with isopropyl ether (20 mL), and dried at approximately 50°C for 8 hours to obtain the product: 28 g of white solid, yield 55%, purity 96.5% (see...). Figure 2 ).

[0053] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing Fmoc-Lys(iPr,Boc)-OH, characterized in that, Includes the following steps: S1, Cbz-Lys-OH and acetone undergo a first-stage reaction under the action of an acidic catalyst; then a second-stage reaction is carried out under the action of a reducing agent; finally, the pH is adjusted to 9-11, Boc2O is added to carry out a third-stage reaction, and Cbz-Lys(iPr,Boc)-OH is obtained by salt formation and purification. S2. Under H2 conditions, the Cbz-Lys(iPr,Boc)-OH described in S1 is hydrogenated in an alcohol solvent under the action of a metal catalyst; then the pH is adjusted to 8-10, Fmoc-OSu is added to carry out the Fmoc protection reaction, and the Fmoc-Lys(iPr,Boc)-OH is obtained by salting and crystallization purification. The salt formation purification involves adding dicyclohexylamine to a solution containing Cbz-Lys(iPr,Boc)-OH or a solution containing Fmoc-Lys(iPr,Boc)-OH to generate the dicyclohexylamine salt of Cbz-Lys(iPr,Boc)-OH or the dicyclohexylamine salt of Fmoc-Lys(iPr,Boc)-OH, which precipitates out as a solid.

2. The method for preparing Fmoc-Lys(iPr,Boc)-OH according to claim 1, characterized in that, In S1, the reducing agent is selected from one or more of sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, and potassium borohydride.

3. The method for preparing Fmoc-Lys(iPr,Boc)-OH according to claim 1, characterized in that, In S1, the molar ratio of Cbz-Lys-OH to Boc2O is 1:(1.0-2.0).

4. The method for preparing Fmoc-Lys(iPr,Boc)-OH according to claim 1, characterized in that, In S1, the reaction time of the first stage is 4h-6h; the reaction time of the second stage is 2h-4h; and the reaction time of the third stage is 3h-5h.

5. The method for preparing Fmoc-Lys(iPr,Boc)-OH according to claim 1, characterized in that, In S2, the metal catalyst is selected from one or more of Pd / C, Pd(OH)2 / C, and Raney nickel.

6. The method for preparing Fmoc-Lys(iPr,Boc)-OH according to claim 1, characterized in that, In S2, the alcohol solvent is selected from one or more of methanol, ethanol, isopropanol and tert-butanol.

7. The method for preparing Fmoc-Lys(iPr,Boc)-OH according to claim 1, characterized in that, In S2, the hydrogenation reaction is carried out at a temperature of 40℃-60℃, a pressure of 0.2MPa-0.4MPa, and a time of 4h-6h.

8. The method for preparing Fmoc-Lys(iPr,Boc)-OH according to claim 1, characterized in that, In S2, the duration of the Fmoc protection reaction is 2-4 hours.

9. The method for preparing Fmoc-Lys(iPr,Boc)-OH according to claim 1, characterized in that, The molar ratio of Cbz-Lys(iPr,Boc)-OH to Fmoc-OSu is 1:(0.9-1.5).

10. The method for preparing Fmoc-Lys(iPr,Boc)-OH according to claim 1, characterized in that, pH adjusters are one or more of potassium carbonate, sodium carbonate, sodium bicarbonate, and potassium bicarbonate.

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