WO2021026800A1 - Method for synthesizing degarelix acetate - Google Patents

Method for synthesizing degarelix acetate Download PDF

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WO2021026800A1
WO2021026800A1 PCT/CN2019/100530 CN2019100530W WO2021026800A1 WO 2021026800 A1 WO2021026800 A1 WO 2021026800A1 CN 2019100530 W CN2019100530 W CN 2019100530W WO 2021026800 A1 WO2021026800 A1 WO 2021026800A1
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fmoc
resin
condensation
fragment
tetrapeptide
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PCT/CN2019/100530
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French (fr)
Chinese (zh)
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李九远
李常峰
李邦洪
朱自力
李新勇
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凯莱英生命科学技术(天津)有限公司
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Priority to PCT/CN2019/100530 priority Critical patent/WO2021026800A1/en
Publication of WO2021026800A1 publication Critical patent/WO2021026800A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides

Definitions

  • the present invention relates to the technical field of organic synthesis, in particular to a method for synthesizing degarelix acetate.
  • Degarelix acetate is one of the main drugs for the treatment of prostate cancer. At present, degarelix is mainly obtained by solid phase synthesis, and then acetic acid is converted into salt to form degarelix acetate.
  • the structure of degarelix is Ac-D-2Nal-D-Phe(4-Cl)-D-Ala(3-Pyridy)-Ser-Aph(Hor)-D-Aph(Cbm)-Leu-Lys(iPr) -Pro-D-Ala-NH 2 , the chemical formula is as follows:
  • the traditional solid-phase synthesis method of degarelix is as follows: A solid-phase synthesis carrier is used to synthesize amino acids one by one, such as patent CN102428097B, CN105085634A and so on. However, in this method, Aph(Hor) is repeatedly contacted with alkali, resulting in partial conversion of dihydrouracil to hydantoin, leading to side reactions.
  • reaction temperature is not properly controlled, acetylation on the side chain Cbm gene is likely to be produced during the final acetylation end-capping process.
  • Chemical by-products which are difficult to separate and purify, will affect the efficiency of the process and the quality of the products.
  • Fmoc-D-4-Aph(t-Bu-Cbm) whose side chain is protected with tert-butyl, the protective group needs to be removed after the connection is completed, and the tert-butyl on the side chain of Aph(t-Bu-Cbm) is removed. It is difficult to remove, and it needs to be completely removed under heating in a strong acid aqueous solution, which increases the possibility of racemization and other impurities and affects product quality.
  • the amino acid residue D-4-Aph(Cbm) at position 6 is synthesized by Fmoc-D-4-Aph(Dde)-OH, and finally capped with acetic anhydride with 2
  • the DMF mixed solution of% hydrazine hydrate removes the Dde on the 6-position D-phenylalanine side chain amino group, and then introduces Cbm through the trimethylsilyl isocyanate DMF solution to avoid the Cbm group in the acetic anhydride blocking process By-products of acetylation.
  • this process is complicated to operate and is not suitable for large-scale production.
  • the literature also reported the Boc solid-phase synthesis method (2001:880SYNTHLINE) and Boc liquid-phase synthesis method (US6214798B1) of degarelix.
  • the Boc solid-phase synthesis method requires HF for cracking, which will cause great harm to the human body and the environment.
  • the purity of degarelix obtained by the Boc liquid phase synthesis method is low, only 96-98% (J. Med. Chem., 2005, 48, 4851).
  • Patent CN109575109A adopts 6+4 solid phase fragment synthesis, the 6-position Aph is first protected with MMt, then removed with 1 to 5% of TFA, and then coupled with L-Hor-OH. However, in the process of removing MMt with TFA, the Ilys side chain Boc will be removed, and then L-Hor-OH will be connected to form impurities, which reduces the yield of the product and is difficult to separate.
  • the patent CN103180335 adopts 3+4+3 and the patent CN106589071 adopts the 6+4 liquid-phase fragment condensation method for synthesis, wherein each fragment is synthesized in the liquid phase.
  • each step of the coupling reaction involves cumbersome N-terminal and C-terminal protection and deprotection processes, as well as the coordination of suitable pH conditions, this has produced a lot of work, and the amount of waste liquid is also large and costly. long time.
  • the efficiency of liquid phase synthesis of peptide fragments is much lower than that of solid phase synthesis of peptide fragments, and separation is also very difficult.
  • the main purpose of the present invention is to provide a method for synthesizing degarelix acetate to solve the problems of multiple side reactions, low yield, high cost and the like when preparing degarelix acetate by solid phase synthesis in the prior art.
  • a method for synthesizing degarelix acetate which includes: S1, using Rink Amide MBHA resin as a solid-phase synthesis carrier, and combining it with the following amino acid sequence in sequence Six protected amino acids Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-iLys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Aph(Cbm)-OH and Fmoc-Aph(Hor) -OH conducts condensation reaction to obtain hexapeptide resin fragment III; S2, CTC resin is used as a solid-phase synthesis carrier, and the following four protected amino acids Fmoc-Ser(tBu)-OH, Fmoc-3- (3-pyridy)-D-Ala-OH, Fmoc-D-Phe(4-Cl)-OH and Fmoc-D
  • step S1 includes: S11, after sequentially swelling the Rink Amide MBHA resin and deprotecting Fmoc, it is condensed with Fmoc-D-Ala-OH for the first time to obtain the first condensation product; S12, the first condensation product is obtained; After the condensation product is deprotected by Fmoc, it is condensed for the second time with Fmoc-D-Ala-OH to obtain the second condensation product; S13, after the second condensation product is Fmoc deprotected, it is combined with Fmoc-iLys( Boc)-OH undergoes the third condensation to obtain the third condensation product; S14, after the third condensation product is Fmoc deprotected, it is subjected to the fourth condensation with Fmoc-Leu-OH to obtain the fourth condensation product; S15 After deprotecting the fourth condensation product by Fmoc, it is condensed with Fmoc-D-Aph(Cbm)-OH for the fifth time to obtain the fifth condensation product
  • step S2 includes: S21, after swelling the CTC resin, it is condensed with Fmoc-Ser(tBu)-OH for the seventh time to obtain a seventh condensation product; S22, after the seventh condensation product is subjected to Fmoc deprotection, It is condensed with Fmoc-3-(3-pyridy)-D-Ala-OH for the eighth time to obtain the eighth condensation product; S23, after the eighth condensation product is Fmoc deprotected, it is combined with Fmoc-D- Phe(4-Cl)-OH undergoes the ninth condensation to obtain the ninth condensation product; S24, after the ninth condensation product is Fmoc deprotected, it is subjected to the tenth condensation with Fmoc-D-2Nal-OH to obtain The tenth condensation product is the tetrapeptide resin fragment I.
  • the condensation system used is independently selected from any one of the following: HOBT/DIC, HOAT/DIC, Oxymapure/DIC, HATU/DIPEA, HBTU/DIPEA, TBTU/DIPEA, PyBOP/DIPEA; the seventh condensation step is carried out in an alkaline environment, and the base used is DIPEA; preferably, each Fmoc deprotection step is used
  • the deprotection reagents are all 20wt% piperidine in DMF solution.
  • the reaction solvent used in each condensation reaction process is one or more of DMF, DCM, and THF.
  • step S1 and step S2 the reaction temperature during each condensation reaction process is 10 to 35° C., and the reaction time is 0.5 to 4 h.
  • step S1 and step S2 during each condensation reaction process, the molar ratio between the protected amino acid, the condensation system and the Fmoc amino resin is 1.5 to 3: 1.5 to 6:1, wherein the moles of the Fmoc amino resin are Contains the number of moles of Fmoc protected amino groups.
  • the degree of substitution of Rink Amide MBHA resin is 0.6-1.0 mmol/g, and the degree of substitution of CTC resin is 0.9-1.6 mmol/g.
  • step S3 also includes the steps of sequentially performing Fmoc deprotection and end-capping reactions on the tetrapeptide resin fragment I, specifically as follows: 20wt% piperidine in DMF solution The tetrapeptide resin fragment I is deprotected by Fmoc to obtain the deprotected fragment I; the deprotected fragment I is subjected to a capping reaction under the action of acetic anhydride and DIPEA to obtain the capped tetrapeptide resin fragment I.
  • step S4 includes: S41, Fmoc deprotection of hexapeptide resin fragment III to obtain deprotected fragment III; S42, mixing tetrapeptide fragment II with a condensing agent and a reaction solvent to obtain a pretreatment liquid; S43, deprotection The treatment liquid undergoes condensation reaction with the deprotected fragment III to obtain the whole peptide resin.
  • reaction solvent used in step S42 is one or more of DMF, DCM, and THF; relative to the hexapeptide resin fragment III, the equivalent of the tetrapeptide fragment II is 1.2 to 1.5 eq, and the condensation agent MYMsA and/or The equivalent of MYTsA is 1.2 ⁇ 1.5eq.
  • step S43 further includes the steps of washing, shrinking, and nitrogen purging and drying the whole peptide resin.
  • step S5 includes: using the cutting fluid to react with the whole peptide resin, and filtering to obtain a filtrate; wherein the cutting fluid is a 95 vol% TFA aqueous solution; using methyl tert-butyl ether to force the filtrate to crystallize and filter to obtain degare Grams of crude product; the crude product is purified to obtain degarelix.
  • step S6 includes: using an aqueous solution of acetic acid to transform degarelix to obtain degarelix acetate.
  • the synthesis method of degarelix acetate provided by the present invention adopts the solid-phase synthesis 6+4 fragment synthesis method.
  • the Rink Amide MBHA resin is used as the solid-phase synthesis carrier to sequentially condense the first six amino acids to form the hexapeptide resin fragment III , And then use CTC resin as a solid-phase synthesis carrier to condense the last four amino acids in sequence to form tetrapeptide resin fragment I, then cut tetrapeptide resin fragment I to obtain tetrapeptide fragment II, and then combine hexapeptide resin fragment III and tetrapeptide fragment II It is condensed to form a whole peptide resin, and finally cut and salted to obtain degarelix acetate.
  • the present invention adopts a solid-phase synthesis 6+4 fragment synthesis method, which is equivalent to Aph(Hor) and alkali only once during the condensation process. Contact, compared to condensing amino acids one by one and repeatedly contacting with alkali, effectively reduces the occurrence of side reactions. Moreover, the present invention uses the condensing agent MYMsA and/or MYTsA in the condensation process of the hexapeptide resin fragment III and the tetrapeptide fragment II.
  • Figure 1 shows the HPLC test results of degarelix acetate prepared according to Example 1 of the present invention.
  • Figure 2 shows the mass spectrometry test results of degarelix acetate prepared according to Example 1 of the present invention.
  • the solid-phase synthesis method in the prior art often has the problems of many side reactions, low yield and high cost when preparing degarelix.
  • the present invention provides a synthetic method of degarelix acetate, which includes: S1, using Rink Amide MBHA resin as a solid-phase synthesis carrier, and combining it with the following six protected amino acids in sequence of amino acids Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-iLys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Aph(Cbm)-OH and Fmoc-Aph(Hor)-OH for condensation After the reaction, the hexapeptide resin fragment III; S2, CTC resin is used as a solid-phase synthesis carrier, and the following four protective amino acids Fmoc-Ser(tBu)-OH, Fmoc-3-(3-pyri
  • the synthesis method of degarelix acetate provided by the present invention adopts the solid-phase synthesis 6+4 fragment synthesis method.
  • the Rink Amide MBHA resin is used as the solid-phase synthesis carrier to sequentially condense the first six amino acids to form the hexapeptide resin fragment III , And then use CTC resin as a solid-phase synthesis carrier to condense the last four amino acids in sequence to form tetrapeptide resin fragment I, and then cut the tetrapeptide resin fragment I to obtain tetrapeptide fragment II (using CTC resin as a solid-phase synthesis carrier, and finally A tetrapeptide fragment II) with a purity of 98-99% is obtained, and then the hexapeptide resin fragment III and the tetrapeptide fragment II are condensed to form a whole peptide resin, and finally degarelix acetate is obtained by cutting and salting.
  • the present invention adopts a solid-phase synthesis 6+4 fragment synthesis method, which is equivalent to Aph(Hor) and alkali only once during the condensation process.
  • Contact after Aph(Hor) condensation only needs to be deprotected in an alkaline environment, compared to condensing amino acids one by one and repeatedly contacting with alkali multiple times, it effectively reduces the occurrence of side reactions.
  • the present invention uses the condensing agent MYMsA and/or MYTsA in the condensation process of the hexapeptide resin fragment III and the tetrapeptide fragment II.
  • the amino acid Aph (Hor) side chain rearrangement impurities is less than 0.1%, the purity of the crude peptide can reach 93-95%, and the purity after purification can reach more than 99.94%, and the total yield Can reach more than 80%.
  • the post-processing operation of this route is simple, and the two segments can be produced at the same time and then assembled into the final product, which improves the efficiency of production amplification. Can be applied to industrial production.
  • the above step S1 includes: S11, after the Rink Amide MBHA resin is swollen and Fmoc deprotected in sequence, it is subjected to the first condensation with Fmoc-D-Ala-OH to obtain the first condensation Product; S12, after the first condensation product is Fmoc deprotected, it is condensed with Fmoc-D-Ala-OH for the second time to obtain the second condensation product; S13, after the second condensation product is Fmoc deprotected, Condense it with Fmoc-iLys(Boc)-OH for the third time to obtain the third condensation product; S14, after deprotecting the third condensation product by Fmoc, it is condensed with Fmoc-Leu-OH for the fourth time, Obtain the fourth condensation product; S15, after the fourth condensation product is Fmoc deprotected, it is condensed with Fmoc-D-Aph(Cbm)-OH for the fifth
  • the above-mentioned step S2 includes: S21, after swelling the CTC resin (the CTC resin does not need to be deprotected, it can directly react with amino acids in an alkaline environment, and a molecule of HCl generated by the reaction is neutralized with alkali ), and Fmoc-Ser(tBu)-OH for the seventh condensation to obtain the seventh condensation product; S22, after the seventh condensation product is Fmoc deprotected, it is combined with Fmoc-3-(3-pyridy)-D -Ala-OH conducts the eighth condensation to obtain the eighth condensation product; S23, after the eighth condensation product is Fmoc deprotected, it is subjected to the ninth condensation with Fmoc-D-Phe(4-Cl)-OH, The ninth condensation product is obtained; S24, after the ninth condensation product is Fmoc deprotected, it is condensed with Fmoc-D-2Nal-OH for the tenth time to obtain
  • the condensation systems used in the first condensation to the sixth condensation step and the eighth condensation to the tenth condensation step are respectively Independently selected from any of the following: HOBT/DIC, HOAT/DIC, Oxymapure/DIC, HATU/DIPEA, HBTU/DIPEA, TBTU/DIPEA, PyBOP/DIPEA; the seventh condensation step is carried out in an alkaline environment, and The base used is DIPEA. (DIPEA only provides an alkaline environment to capture the removed HCl, no condensation agent is needed in this step).
  • DIC is the activator
  • HOBT HOAT
  • Oxymapure are racemization inhibitors
  • DIPEA is the base
  • HATU, HBTU, TBTU, and PyBoP are the activators.
  • the deprotection reagent used in each Fmoc deprotection step is a DMF solution of 20 wt% piperidine.
  • the reaction solvent used in each condensation reaction process is one or more of DMF, DCM, and THF.
  • the above-mentioned solvents have good solubility for amino acids and can provide a more stable environment for the condensation reaction.
  • the reaction temperature during each condensation reaction process is 10 to 35° C., and the reaction time is 0.5 to 4 h.
  • the molar ratio between the protected amino acid, the condensation system and the Fmoc amino resin is 1.5 to 3: 1.5 to 6:1, where Fmoc
  • the number of moles of amino resin is based on the number of moles of Fmoc protected amino group contained.
  • the protected amino acid here refers to the Fmoc protected amino acid during each amino acid condensation reaction, such as Fmoc-D-Ala-OH in the first condensation step, and Fmoc-D-Ala-OH in the second condensation step.
  • Fmoc amino resin refers to the resin side in each condensation reaction process, such as Rink Amide MBHA resin in the first condensation step, the first condensation product in the second condensation step, and the number of moles of Fmoc amino resin is The number of moles of Fmoc protected amino groups is equivalent to the number of sites that can be reacted. Controlling the molar ratio between the protected amino acid, the condensation system and the Fmoc amino resin within the above range not only helps to improve the reaction efficiency and conversion rate, but also reduces the waste of raw materials.
  • the degree of substitution of Rink Amide MBHA resin is 0.6-1.0 mmol/g, and the substitution of CTC resin The degree is 0.9 ⁇ 1.6mmol/g.
  • step S3 further includes the steps of sequentially performing Fmoc deprotection and end-capping reactions on the tetrapeptide resin fragment I, specifically as follows: 20wt%
  • the DMF solution of piperidine performs Fmoc deprotection on the tetrapeptide resin fragment I to obtain the deprotected fragment I; the deprotected fragment I is subjected to a capping reaction under the action of acetic anhydride and DIPEA to obtain the capped tetrapeptide resin fragment I .
  • the amino groups in the tetrapeptide resin fragment I that are not involved in the condensation reaction can be capped to prevent them from participating in subsequent reactions, thereby further reducing the occurrence of side reactions.
  • the above step S4 includes: S41, Fmoc deprotection of the hexapeptide resin fragment III to obtain the deprotected fragment III; S42, mixing the tetrapeptide fragment II with the condensing agent and the reaction solvent to obtain the pre Treatment solution; S43, the pretreatment solution and the deprotected fragment III are subjected to a condensation reaction to obtain a whole peptide resin.
  • S41 Fmoc deprotection of the hexapeptide resin fragment III to obtain the deprotected fragment III
  • S42 mixing the tetrapeptide fragment II with the condensing agent and the reaction solvent to obtain the pre Treatment solution
  • S43 the pretreatment solution and the deprotected fragment III are subjected to a condensation reaction to obtain a whole peptide resin.
  • Fmoc is deprotected
  • the terminal amino group of the hexapeptide resin fragment III is exposed, and then it undergoes a condensation reaction with the terminal hydroxyl group of the t
  • the reaction solvent used in step S42 is one or more of DMF, DCM, and THF; compared to the hexapeptide resin fragment III, the equivalent of the tetrapeptide fragment II is 1.2-1.5 eq, and the equivalent of the condensing agent MYMsA and/or MYTsA is 1.2-1.5 eq. More preferably, after the whole peptide resin is obtained, step S43 further includes the steps of washing, shrinking, and nitrogen purging and drying the whole peptide resin.
  • the above step S5 includes: after the cleavage fluid is reacted with the whole peptide resin, filtered to obtain the filtrate; wherein the cleavage fluid is a 95 vol% TFA aqueous solution; and methyl tert-butyl ether is used to force the filtrate to crystallize , Filter to obtain the crude product of degarelix; purify the crude product to obtain degarelix.
  • the linear peptide chains in the whole peptide resin can be completely separated.
  • the above purification process adopts reverse high performance liquid chromatography, specifically using AQ packing, phase A adopts 0.05wt% TFA aqueous solution, and phase B adopts 0.05wt% TFA acetonitrile solution.
  • the above step S6 includes: using an aqueous solution of acetic acid to salt degarelix to obtain degarelix acetate. Specifically, after purification by reverse-phase high performance liquid chromatography, a 1 wt% AcOH aqueous solution and acetonitrile are used as mobile phase gradient elution and salt conversion, and then freeze-dried to obtain the degarelix acetate product.
  • Example 1 degarelix acetate was synthesized, and the specific process is as follows:
  • the tetrapeptide resin fragment I was cleaved with 0.5wt% TFA/DCM solution for 2h. After the filtrate was concentrated, methyl tert-butyl ether was used to crystallize and filtered to obtain tetrapeptide fragment II, totaling 66.6 g, with a yield of 95%.
  • the resin is washed and shrunk alternately with methanol and dichloromethane. After the shrinkage is completed, the nitrogen purge and bake the material to a constant weight and then the material is collected to obtain the peptide resin.
  • the connected whole peptide resin is reacted with 95vol%TFA/5vol%H 2 O ratio cutting solution for 3 ⁇ 5h. After the filtrate is concentrated, methyl tert-butyl ether is used to crystallize and filter to obtain 104.3g of the final product. The yield of crude peptide is 94 %, 95% purity.
  • Example 1 The difference from Example 1 is that in each condensation reaction step, the ratio of the amount of the protected amino acid, the corresponding condensation system and the Fmoc amino resin is 3:1.5:1, and the ratio of each component in the condensation system remains unchanged.
  • the yield of the product was 86%, and the purity was 82%.
  • Example 1 The difference from Example 1 is that in each condensation reaction step, the ratio of the amount of the protected amino acid, the corresponding condensation system and the Fmoc amino resin is 1.5:3:1, and the ratio of each component in the condensation system remains unchanged.
  • the yield of the product was 87%, and the purity was 83%.
  • Example 1 The difference from Example 1 is that in each condensation reaction step, the ratio of the amount of the protected amino acid, the corresponding condensation system and the Fmoc amino resin is 1.2:1.2:1, and the ratio of each component in the condensation system remains unchanged.
  • the yield of the product is 85% and the purity is 79%.
  • Example 1 The difference from Example 1 is that in each condensation reaction step of the synthesis process of hexapeptide resin fragment III and tetrapeptide resin fragment II, the corresponding condensation system is DIC/HOBT, and the ratio of other components remains unchanged.
  • the yield of crude peptide is 93 %, the purity is 94%.
  • Example 1 The difference from Example 1 is that in each condensation reaction step of the synthesis process of hexapeptide resin fragment III and tetrapeptide resin fragment II, the corresponding condensation system is HBTU/DIPEA, and the ratio of other components remains unchanged.
  • the yield of crude peptide is 92 %, the purity is 95%.
  • Example 1 The difference from Example 1 is that in each condensation reaction step of the synthesis process of hexapeptide resin fragment III and tetrapeptide resin fragment II, the corresponding condensation system is PyBoP/DIPEA, and the ratio of other components remains unchanged.
  • the yield of crude peptide is 95 %, the purity is 93%.
  • Example 1 The difference from Example 1 is: the whole peptide resin condensation system is HATU/DIPEA, the crude peptide yield is 75%, and the purity is 81%.
  • Example 1 The difference from Example 1 is: the whole peptide resin condensation system is PyBOP/DIPEA. The yield of the crude peptide was 77% and the purity was 79%.
  • Example 2 The difference from Example 1 is that the whole peptide resin condensation system is DIC/HOBT. The yield of the crude peptide was 86% and the purity was 82%.

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Abstract

Disclosed is a method for synthesizing Degarelix acetate. The method comprises: S1, carrying out a condensation reaction with the first six amino acids of Degarelix in sequence by using a Rink Amide MBHA resin as a carrier so as to obtain a hexapeptide resin fragment III; S2, carrying out a condensation reaction with the last four amino acids of Degarelix in sequence by using a CTC resin as a carrier so as to obtain a tetrapeptide resin fragment I; S3, cutting the tetrapeptide resin fragment I to obtain a tetrapeptide fragment II; S4, carrying out a condensation reaction on the hexapeptide resin fragment III and the tetrapeptide fragment II under the action of MYMsA and/or MYTsA so as to obtain a whole-peptide resin; S5, cutting the whole-peptide resin to obtain Degarelix; and S6, carrying out acetic acid salt conversion on the Degarelix to obtain Degarelix acetate.

Description

醋酸地加瑞克的合成方法Synthesis method of degarelix acetate 技术领域Technical field
本发明涉及有机合成技术领域，具体而言，涉及一种醋酸地加瑞克的合成方法。The present invention relates to the technical field of organic synthesis, in particular to a method for synthesizing degarelix acetate.
背景技术Background technique
醋酸地加瑞克(Degarelix acetate)是治疗***癌的主要用药之一，目前主要通过固相合成得到地加瑞克，然后进行醋酸转盐形成醋酸地加瑞克。地加瑞克结构为Ac-D-2Nal-D-Phe(4-Cl)-D-Ala(3-Pyridy)-Ser-Aph(Hor)-D-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH 2，化学式如下：
Figure PCTCN2019100530-appb-000001
Degarelix acetate is one of the main drugs for the treatment of prostate cancer. At present, degarelix is mainly obtained by solid phase synthesis, and then acetic acid is converted into salt to form degarelix acetate. The structure of degarelix is Ac-D-2Nal-D-Phe(4-Cl)-D-Ala(3-Pyridy)-Ser-Aph(Hor)-D-Aph(Cbm)-Leu-Lys(iPr) -Pro-D-Ala-NH 2 , the chemical formula is as follows:
Figure PCTCN2019100530-appb-000001
地加瑞克的传统固相合成方法如下：通过固相合成载体进行逐个的氨基酸合成，比如专利CN102428097B，CN105085634A等。然而，该方法中Aph(Hor)重复与碱接触，导致二氢尿嘧啶部分转化为乙内酰，导致副反应发生。The traditional solid-phase synthesis method of degarelix is as follows: A solid-phase synthesis carrier is used to synthesize amino acids one by one, such as patent CN102428097B, CN105085634A and so on. However, in this method, Aph(Hor) is repeatedly contacted with alkali, resulting in partial conversion of dihydrouracil to hydantoin, leading to side reactions.
专利CN102329373A,CN103992392A,CN10292174A,CN102428097B等公开的地加瑞克合成方法中，6位氨基酸残基D-4-Aph(Cbm)在合成中采用了侧链Cbm基团不保护的Fmoc-D-4-Aph(Cbm)-OH或者侧链Cbm用t-Bu保护的Fmoc-D-4-Aph(t-Bu-Cbm)-OH。然而，固相合成中采用侧链不保护的Fmoc-D-4-Aph(Cbm)-OH，如果反应温度控制不当，在最后的乙酰化封端过程中容易产生在侧链Cbm基因上的乙酰化副产物，该副产物难以分离纯化，将影响工艺的效率和产品的质量。采用侧链用叔丁基保护的Fmoc-D-4-Aph(t-Bu-Cbm),在完成连结后需要去除保护基，而Aph(t-Bu-Cbm)侧链上叔丁基的脱除比较困难，需要在强酸水溶液中加热条件下才能完全脱除，加大了产生消旋等杂质的可能性，影响产品质量。In patents CN102329373A, CN103992392A, CN10292174A, CN102428097B and other disclosed degarelix synthesis methods, the amino acid residue D-4-Aph (Cbm) at position 6 is synthesized using Fmoc-D-4 whose side chain Cbm group is not protected. -Aph(Cbm)-OH or Fmoc-D-4-Aph(t-Bu-Cbm)-OH whose side chain Cbm is protected with t-Bu. However, Fmoc-D-4-Aph(Cbm)-OH with unprotected side chain is used in solid-phase synthesis. If the reaction temperature is not properly controlled, acetylation on the side chain Cbm gene is likely to be produced during the final acetylation end-capping process. Chemical by-products, which are difficult to separate and purify, will affect the efficiency of the process and the quality of the products. Using Fmoc-D-4-Aph(t-Bu-Cbm) whose side chain is protected with tert-butyl, the protective group needs to be removed after the connection is completed, and the tert-butyl on the side chain of Aph(t-Bu-Cbm) is removed. It is difficult to remove, and it needs to be completely removed under heating in a strong acid aqueous solution, which increases the possibility of racemization and other impurities and affects product quality.
专利CN103992378A公开的地加瑞克合成方法中，6位氨基酸残基D-4-Aph(Cbm)在合成中采用Fmoc-D-4-Aph(Dde)-OH,最后醋酐封端后用2％水合肼的DMF混合溶液脱去6位D-苯丙氨酸侧链氨基上的Dde，再通过三甲基硅基异氰酸酯DMF溶液引入Cbm，以此来避免醋酐封端过程中Cbm基团的乙酰化副产物。然而，此工艺操作复杂，不适于规模化生产。In the synthetic method of degarelix disclosed in the patent CN103992378A, the amino acid residue D-4-Aph(Cbm) at position 6 is synthesized by Fmoc-D-4-Aph(Dde)-OH, and finally capped with acetic anhydride with 2 The DMF mixed solution of% hydrazine hydrate removes the Dde on the 6-position D-phenylalanine side chain amino group, and then introduces Cbm through the trimethylsilyl isocyanate DMF solution to avoid the Cbm group in the acetic anhydride blocking process By-products of acetylation. However, this process is complicated to operate and is not suitable for large-scale production.
文献也报道了地加瑞克的Boc固相合成法(2001:880SYNTHLINE)和Boc液相合成法(US6214798B1)。Boc固相合成法需要用HF进行裂解，对人体和环境都会造成很大危害。Boc液相合成法所得地加瑞克纯度较低，只有96～98％(J.Med.Chem.,2005,48,4851)。The literature also reported the Boc solid-phase synthesis method (2001:880SYNTHLINE) and Boc liquid-phase synthesis method (US6214798B1) of degarelix. The Boc solid-phase synthesis method requires HF for cracking, which will cause great harm to the human body and the environment. The purity of degarelix obtained by the Boc liquid phase synthesis method is low, only 96-98% (J. Med. Chem., 2005, 48, 4851).
专利CN109575109A采用6+4的固相片段合成，6位Aph先用MMt保护，然后用1～5％的TFA脱除后，在用L-Hor-OH偶联。然而，在用TFA脱MMt过程中，会使ILys侧链Boc脱除，然后L-Hor-OH连接上，生成杂质，降低了产品的收率，且分离难度很大。Patent CN109575109A adopts 6+4 solid phase fragment synthesis, the 6-position Aph is first protected with MMt, then removed with 1 to 5% of TFA, and then coupled with L-Hor-OH. However, in the process of removing MMt with TFA, the Ilys side chain Boc will be removed, and then L-Hor-OH will be connected to form impurities, which reduces the yield of the product and is difficult to separate.
另外还有一些固相片段合成方法用以合成地加瑞克，比如专利CN103351428和专利CN107022002采用固相片段缩合的方法合成。然而，固相片段缩合投入的每个片段都是2倍过量，严重浪费肽片段，造成合成成本过高。同时，因固相片段缩合的树脂取代值限制，物料通量降低，浪费溶剂，产生大量废液。In addition, there are some solid-phase fragment synthesis methods for synthesizing degarelix, such as patent CN103351428 and patent CN107022002 using solid-phase fragment condensation methods. However, each fragment invested in solid-phase fragment condensation is a 2-fold excess, which seriously wastes peptide fragments and causes excessive synthesis costs. At the same time, due to the limitation of the resin substitution value of the solid phase fragment condensation, the material flux is reduced, the solvent is wasted, and a large amount of waste liquid is generated.
除了固相合成方法，专利CN103180335采用3+4+3、专利CN106589071采用6+4的液相片段缩合的方法合成，其中每个片段均是液相合成。然而，因每一步偶合反应都涉及了繁琐的N末端以及C末端的保护和脱保护过程，以及适宜的pH条件的配合，这产生了很大的工作量，废液量也很大，且耗费时间很长。而且液相合成肽片段的效率远低于固相合成肽片段，分离也有很大难度。In addition to the solid-phase synthesis method, the patent CN103180335 adopts 3+4+3 and the patent CN106589071 adopts the 6+4 liquid-phase fragment condensation method for synthesis, wherein each fragment is synthesized in the liquid phase. However, because each step of the coupling reaction involves cumbersome N-terminal and C-terminal protection and deprotection processes, as well as the coordination of suitable pH conditions, this has produced a lot of work, and the amount of waste liquid is also large and costly. long time. Moreover, the efficiency of liquid phase synthesis of peptide fragments is much lower than that of solid phase synthesis of peptide fragments, and separation is also very difficult.
发明内容Summary of the invention
本发明的主要目的在于提供一种醋酸地加瑞克的合成方法，以解决现有技术中采用固相合成法制备醋酸地加瑞克时存在的副反应多、收率底、成本高等问题。The main purpose of the present invention is to provide a method for synthesizing degarelix acetate to solve the problems of multiple side reactions, low yield, high cost and the like when preparing degarelix acetate by solid phase synthesis in the prior art.
为了实现上述目的，根据本发明的一个方面，提供了一种醋酸地加瑞克的合成方法，其包括：S1，以Rink Amide MBHA树脂为固相合成的载体，将其按照氨基酸顺序依次与以下六种保护氨基酸Fmoc-D-Ala-OH、Fmoc-Pro-OH、Fmoc-iLys(Boc)-OH、Fmoc-Leu-OH、Fmoc-D-Aph(Cbm)-OH及Fmoc-Aph(Hor)-OH进行缩合反应，得到六肽树脂片段III；S2，以CTC树脂为固相合成的载体，将其按照氨基酸顺序依次与以下四种保护氨基酸Fmoc-Ser(tBu)-OH、Fmoc-3-(3-pyridy)-D-Ala-OH、Fmoc-D-Phe(4-Cl)-OH及Fmoc-D-2Nal-OH进行缩合反应，得到四肽树脂片段I；S3，切割四肽树脂片段I的树脂，得到四肽片段II；S4，将六肽树脂片段III与四肽片段II在缩合剂MYMsA和/或MYTsA的作用下进行缩合反应，得到全肽树脂；S5，切割全肽树脂的树脂，得到地加瑞克；S6，对地加瑞克进行醋酸转盐，得到醋酸地加瑞克。In order to achieve the above objective, according to one aspect of the present invention, a method for synthesizing degarelix acetate is provided, which includes: S1, using Rink Amide MBHA resin as a solid-phase synthesis carrier, and combining it with the following amino acid sequence in sequence Six protected amino acids Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-iLys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Aph(Cbm)-OH and Fmoc-Aph(Hor) -OH conducts condensation reaction to obtain hexapeptide resin fragment III; S2, CTC resin is used as a solid-phase synthesis carrier, and the following four protected amino acids Fmoc-Ser(tBu)-OH, Fmoc-3- (3-pyridy)-D-Ala-OH, Fmoc-D-Phe(4-Cl)-OH and Fmoc-D-2Nal-OH undergo condensation reaction to obtain tetrapeptide resin fragment I; S3, cleavage tetrapeptide resin fragment I resin to obtain the tetrapeptide fragment II; S4, the hexapeptide resin fragment III and the tetrapeptide fragment II are subjected to condensation reaction under the action of the condensation agent MYMsA and/or MYTsA to obtain the whole peptide resin; S5, the whole peptide resin is cut Resin, degarelix is obtained; S6, degarelix is converted into acetic acid to obtain degarelix acetate.
进一步地，步骤S1包括：S11，将Rink Amide MBHA树脂依次进行溶胀、Fmoc脱保护后，将其与Fmoc-D-Ala-OH进行第一次缩合，得到第一缩合产物；S12，将第一缩合产物进行Fmoc脱保护后，将其与Fmoc-D-Ala-OH进行第二次缩合，得到第二缩合产物；S13，将第二缩合产物进行Fmoc脱保护后，将其与Fmoc-iLys(Boc)-OH进行第三次缩合，得到第三缩合产物；S14，将第三缩合产物进行Fmoc脱保护后，将其与Fmoc-Leu-OH进行第四次缩合，得到第四缩合产物；S15，将第四缩合产物进行Fmoc脱保护后，将其与Fmoc-D-Aph(Cbm)-OH进 行第五次缩合，得到第五缩合产物；S16，将第五缩合产物进行Fmoc脱保护后，将其与Fmoc-Aph(Hor)-OH进行第六次缩合，得到第六缩合产物，即为六肽树脂片段III。Further, step S1 includes: S11, after sequentially swelling the Rink Amide MBHA resin and deprotecting Fmoc, it is condensed with Fmoc-D-Ala-OH for the first time to obtain the first condensation product; S12, the first condensation product is obtained; After the condensation product is deprotected by Fmoc, it is condensed for the second time with Fmoc-D-Ala-OH to obtain the second condensation product; S13, after the second condensation product is Fmoc deprotected, it is combined with Fmoc-iLys( Boc)-OH undergoes the third condensation to obtain the third condensation product; S14, after the third condensation product is Fmoc deprotected, it is subjected to the fourth condensation with Fmoc-Leu-OH to obtain the fourth condensation product; S15 After deprotecting the fourth condensation product by Fmoc, it is condensed with Fmoc-D-Aph(Cbm)-OH for the fifth time to obtain the fifth condensation product; S16, after the fifth condensation product is deprotected by Fmoc, It is condensed with Fmoc-Aph(Hor)-OH for the sixth time to obtain the sixth condensation product, which is the hexapeptide resin fragment III.
进一步地，步骤S2包括：S21，将CTC树脂进行溶胀后，与Fmoc-Ser(tBu)-OH进行第七次缩合，得到第七缩合产物；S22，将第七缩合产物进行Fmoc脱保护后，将其与Fmoc-3-(3-pyridy)-D-Ala-OH进行第八次缩合，得到第八缩合产物；S23，将第八缩合产物进行Fmoc脱保护后，将其与Fmoc-D-Phe(4-Cl)-OH进行第九次缩合，得到第九缩合产物；S24，将第九缩合产物进行Fmoc脱保护后，将其与Fmoc-D-2Nal-OH进行第十次缩合，得到第十缩合产物，即为四肽树脂片段I。Further, step S2 includes: S21, after swelling the CTC resin, it is condensed with Fmoc-Ser(tBu)-OH for the seventh time to obtain a seventh condensation product; S22, after the seventh condensation product is subjected to Fmoc deprotection, It is condensed with Fmoc-3-(3-pyridy)-D-Ala-OH for the eighth time to obtain the eighth condensation product; S23, after the eighth condensation product is Fmoc deprotected, it is combined with Fmoc-D- Phe(4-Cl)-OH undergoes the ninth condensation to obtain the ninth condensation product; S24, after the ninth condensation product is Fmoc deprotected, it is subjected to the tenth condensation with Fmoc-D-2Nal-OH to obtain The tenth condensation product is the tetrapeptide resin fragment I.
进一步地，第一次缩合至第六次缩合步骤中，以及第八次缩合至第十次缩合步骤中，采用的缩合体系分别独立地选自以下任一种：HOBT/DIC、HOAT/DIC、Oxymapure/DIC、HATU/DIPEA、HBTU/DIPEA、TBTU/DIPEA、PyBOP/DIPEA；第七次缩合步骤在碱性环境中进行，且采用的碱为DIPEA；优选地，各Fmoc脱保护步骤中采用的脱保护试剂均为20wt％哌啶的DMF溶液。Further, in the first condensation to the sixth condensation step, and in the eighth condensation to the tenth condensation step, the condensation system used is independently selected from any one of the following: HOBT/DIC, HOAT/DIC, Oxymapure/DIC, HATU/DIPEA, HBTU/DIPEA, TBTU/DIPEA, PyBOP/DIPEA; the seventh condensation step is carried out in an alkaline environment, and the base used is DIPEA; preferably, each Fmoc deprotection step is used The deprotection reagents are all 20wt% piperidine in DMF solution.
进一步地，步骤S1和步骤S2中，各缩合反应过程中采用的反应溶剂为DMF、DCM、THF中的一种或多种。Further, in step S1 and step S2, the reaction solvent used in each condensation reaction process is one or more of DMF, DCM, and THF.
进一步地，步骤S1和步骤S2中，各缩合反应过程中的反应温度为10～35℃，反应时间为0.5～4h。Further, in step S1 and step S2, the reaction temperature during each condensation reaction process is 10 to 35° C., and the reaction time is 0.5 to 4 h.
进一步地，步骤S1和步骤S2中，各缩合反应过程中，保护氨基酸、缩合体系及Fmoc氨基树脂之间的摩尔比为1.5～3:1.5～6:1，其中Fmoc氨基树脂的摩尔数以所含Fmoc保护氨基的摩尔数计。Further, in step S1 and step S2, during each condensation reaction process, the molar ratio between the protected amino acid, the condensation system and the Fmoc amino resin is 1.5 to 3: 1.5 to 6:1, wherein the moles of the Fmoc amino resin are Contains the number of moles of Fmoc protected amino groups.
进一步地，Rink Amide MBHA树脂的取代度为0.6～1.0mmol/g，CTC树脂的取代度为0.9～1.6mmol/g。Further, the degree of substitution of Rink Amide MBHA resin is 0.6-1.0 mmol/g, and the degree of substitution of CTC resin is 0.9-1.6 mmol/g.
进一步地，在切割四肽树脂片段I的树脂的步骤之前，步骤S3还包括对四肽树脂片段I依次进行Fmoc脱保护、封端反应的步骤，具体如下：采用20wt％哌啶的DMF溶液对四肽树脂片段I进行Fmoc脱保护，得到脱保护片段I；将脱保护片段I与乙酸酐、DIPEA的作用下进行封端反应，得到封端后的四肽树脂片段I。Further, before the step of cleaving the resin of the tetrapeptide resin fragment I, step S3 also includes the steps of sequentially performing Fmoc deprotection and end-capping reactions on the tetrapeptide resin fragment I, specifically as follows: 20wt% piperidine in DMF solution The tetrapeptide resin fragment I is deprotected by Fmoc to obtain the deprotected fragment I; the deprotected fragment I is subjected to a capping reaction under the action of acetic anhydride and DIPEA to obtain the capped tetrapeptide resin fragment I.
进一步地，步骤S4包括：S41，将六肽树脂片段III进行Fmoc脱保护，得到脱保护片段III；S42，将四肽片段II和缩合剂、反应溶剂混合，得到预处理液；S43，将预处理液与脱保护片段III进行缩合反应，得到全肽树脂。Further, step S4 includes: S41, Fmoc deprotection of hexapeptide resin fragment III to obtain deprotected fragment III; S42, mixing tetrapeptide fragment II with a condensing agent and a reaction solvent to obtain a pretreatment liquid; S43, deprotection The treatment liquid undergoes condensation reaction with the deprotected fragment III to obtain the whole peptide resin.
进一步地，步骤S42中采用的反应溶剂为DMF、DCM、THF中的一种或多种；相对于六肽树脂片段III，四肽片段II的当量为1.2～1.5eq，缩合剂MYMsA和/或MYTsA的当量为1.2～1.5eq。Further, the reaction solvent used in step S42 is one or more of DMF, DCM, and THF; relative to the hexapeptide resin fragment III, the equivalent of the tetrapeptide fragment II is 1.2 to 1.5 eq, and the condensation agent MYMsA and/or The equivalent of MYTsA is 1.2～1.5eq.
进一步地，在得到全肽树脂后，步骤S43还包括对全肽树脂进行洗涤、收缩、氮气吹扫烘干的步骤。Further, after the whole peptide resin is obtained, step S43 further includes the steps of washing, shrinking, and nitrogen purging and drying the whole peptide resin.
进一步地，步骤S5包括：采用切割液与全肽树脂反应后，过滤，得到滤液；其中切割液为95vol％TFA的水溶液；采用甲基叔丁基醚对滤液逼晶，过滤，得到地加瑞克的粗产物；对粗产物进行提纯，得到地加瑞克。Further, step S5 includes: using the cutting fluid to react with the whole peptide resin, and filtering to obtain a filtrate; wherein the cutting fluid is a 95 vol% TFA aqueous solution; using methyl tert-butyl ether to force the filtrate to crystallize and filter to obtain degare Grams of crude product; the crude product is purified to obtain degarelix.
进一步地，步骤S6包括：采用醋酸水溶液对地加瑞克进行转盐，得到醋酸地加瑞克。Further, step S6 includes: using an aqueous solution of acetic acid to transform degarelix to obtain degarelix acetate.
本发明提供的醋酸地加瑞克的合成方法中采用了固相合成6+4片段合成法，先以Rink Amide MBHA树脂为固相合成的载体将前六个氨基酸依次缩合形成六肽树脂片段III，再以CTC树脂为固相合成的载体将后四个氨基酸依次缩合形成四肽树脂片段I，其次切割四肽树脂片段I得到四肽片段II，然后将六肽树脂片段III和四肽片段II缩合形成全肽树脂，最后经切割、转盐得到醋酸地加瑞克。氨基酸中易引发副反应的NH 2-Aph(Hor)-OH位于第六个，本发明采用固相合成6+4片段合成法，相当于在缩合过程中Aph(Hor)只与碱进行了一次接触，相比逐一缩合氨基酸反复与碱进行多次接触，有效减少了副反应的发生。而且，本发明在六肽树脂片段III和四肽片段II的缩合过程中使用了缩合剂MYMsA和/或MYTsA，这两种缩合剂无消旋化影响，缩合能力强，且不需要添加任何辅助剂和催化剂，有利于减少片段的消耗量。以上两方面的原因使得醋酸地加瑞克的合成收率明显提高，副反应明显减少，合成成本也相应降低。 The synthesis method of degarelix acetate provided by the present invention adopts the solid-phase synthesis 6+4 fragment synthesis method. First, the Rink Amide MBHA resin is used as the solid-phase synthesis carrier to sequentially condense the first six amino acids to form the hexapeptide resin fragment III , And then use CTC resin as a solid-phase synthesis carrier to condense the last four amino acids in sequence to form tetrapeptide resin fragment I, then cut tetrapeptide resin fragment I to obtain tetrapeptide fragment II, and then combine hexapeptide resin fragment III and tetrapeptide fragment II It is condensed to form a whole peptide resin, and finally cut and salted to obtain degarelix acetate. Among amino acids, NH 2 -Aph(Hor)-OH, which is prone to cause side reactions, is located at the sixth place. The present invention adopts a solid-phase synthesis 6+4 fragment synthesis method, which is equivalent to Aph(Hor) and alkali only once during the condensation process. Contact, compared to condensing amino acids one by one and repeatedly contacting with alkali, effectively reduces the occurrence of side reactions. Moreover, the present invention uses the condensing agent MYMsA and/or MYTsA in the condensation process of the hexapeptide resin fragment III and the tetrapeptide fragment II. These two condensing agents have no racemization effect, strong condensation ability, and do not need to add any auxiliary Agents and catalysts help reduce the consumption of fragments. The above two reasons make the synthesis yield of degarelix acetate significantly improved, side reactions are significantly reduced, and the synthesis cost is correspondingly reduced.
附图说明Description of the drawings
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：The accompanying drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments and descriptions of the present invention are used to explain the present invention, and do not constitute an improper limitation of the present invention. In the attached picture:
图1示出了根据本发明实施例1制备的醋酸地加瑞克的HPLC测试结果；以及Figure 1 shows the HPLC test results of degarelix acetate prepared according to Example 1 of the present invention; and
图2示出了根据本发明实施例1制备的醋酸地加瑞克的质谱测试结果。Figure 2 shows the mass spectrometry test results of degarelix acetate prepared according to Example 1 of the present invention.
具体实施方式detailed description
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。It should be noted that the embodiments in this application and the features in the embodiments can be combined with each other if there is no conflict. Hereinafter, the present invention will be described in detail with reference to the drawings and in conjunction with the embodiments.
如本发明背景技术部分所描述的，现有技术中采用固相合成法制备地加瑞克时多存在副反应多、收率低、成本高的问题。为了解决这一问题，本发明提供了一种醋酸地加瑞克的合成方法，其包括：S1，以Rink Amide MBHA树脂为固相合成的载体，将其按照氨基酸顺序依次与以下六种保护氨基酸Fmoc-D-Ala-OH、Fmoc-Pro-OH、Fmoc-iLys(Boc)-OH、Fmoc-Leu-OH、Fmoc-D-Aph(Cbm)-OH及Fmoc-Aph(Hor)-OH进行缩合反应，得到六肽树脂片段III；S2，以CTC树脂为固相合成的载体，将其按照氨基酸顺序依次与以下四种保护氨基酸Fmoc-Ser(tBu)-OH、Fmoc-3-(3-pyridy)-D-Ala-OH、Fmoc-D-Phe(4-Cl)-OH及Fmoc-D-2Nal-OH 进行缩合反应，得到四肽树脂片段I；S3，切割四肽树脂片段I的树脂，得到四肽片段II；S4，将六肽树脂片段III与四肽片段II在缩合剂MYMsA和/或MYTsA的作用下进行缩合反应，得到全肽树脂；S5，切割全肽树脂的树脂，得到地加瑞克；S6，对地加瑞克进行醋酸转盐，得到醋酸地加瑞克。As described in the background art part of the present invention, the solid-phase synthesis method in the prior art often has the problems of many side reactions, low yield and high cost when preparing degarelix. In order to solve this problem, the present invention provides a synthetic method of degarelix acetate, which includes: S1, using Rink Amide MBHA resin as a solid-phase synthesis carrier, and combining it with the following six protected amino acids in sequence of amino acids Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-iLys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Aph(Cbm)-OH and Fmoc-Aph(Hor)-OH for condensation After the reaction, the hexapeptide resin fragment III; S2, CTC resin is used as a solid-phase synthesis carrier, and the following four protective amino acids Fmoc-Ser(tBu)-OH, Fmoc-3-(3-pyridy )-D-Ala-OH, Fmoc-D-Phe(4-Cl)-OH and Fmoc-D-2Nal-OH undergo condensation reactions to obtain the tetrapeptide resin fragment I; S3, the resin for cutting the tetrapeptide resin fragment I, Obtain the tetrapeptide fragment II; S4, the hexapeptide resin fragment III and the tetrapeptide fragment II are subjected to condensation reaction under the action of the condensation agent MYMsA and/or MYTsA to obtain the whole peptide resin; S5, the resin of the whole peptide resin is cut to obtain the ground Garrick; S6, acetic acid transsalting of degarelix to obtain degarelix acetate.
四肽树脂片段I、四肽片段II、六肽树脂片段III、全肽树脂Ⅳ、地加瑞克Ⅴ的结构如下：The structures of tetrapeptide resin fragment I, tetrapeptide fragment II, hexapeptide resin fragment III, whole peptide resin IV, and degarelix V are as follows:
Figure PCTCN2019100530-appb-000002
Figure PCTCN2019100530-appb-000002
本发明提供的醋酸地加瑞克的合成方法中采用了固相合成6+4片段合成法，先以Rink Amide MBHA树脂为固相合成的载体将前六个氨基酸依次缩合形成六肽树脂片段III，再以CTC树脂为固相合成的载体将后四个氨基酸依次缩合形成四肽树脂片段I，其次切割四肽树脂片段I得到四肽片段II(采用CTC树脂为固相合成的载体，最终可得到纯度达98～99％的四肽片段II)，然后将六肽树脂片段III和四肽片段II缩合形成全肽树脂，最后经切割、转盐得到醋酸地加瑞克。氨基酸中易引发副反应的NH 2-Aph(Hor)-OH位于第六个，本发明采用固相合成6+4片段合成法，相当于在缩合过程中Aph(Hor)只与碱进行了一次接触(在Aph(Hor)缩合后只需在碱性环境中进行脱保护)，相比逐一缩合氨基酸反复与碱进行多次接触，有效减少了副反应的发生。而且，本发明在六肽树脂片段III和四肽片段II的缩合过程中使用了缩合剂MYMsA和/或MYTsA，这两种缩合剂无消旋化影响，缩合能力强，且不需要添加任何辅助剂和催化剂，有利于减少片段的消耗量。以上两方面的原因使得醋酸地加瑞克的合成收率明显提高，副反应明显减少，合成成本也相应降低。 The synthesis method of degarelix acetate provided by the present invention adopts the solid-phase synthesis 6+4 fragment synthesis method. First, the Rink Amide MBHA resin is used as the solid-phase synthesis carrier to sequentially condense the first six amino acids to form the hexapeptide resin fragment III , And then use CTC resin as a solid-phase synthesis carrier to condense the last four amino acids in sequence to form tetrapeptide resin fragment I, and then cut the tetrapeptide resin fragment I to obtain tetrapeptide fragment II (using CTC resin as a solid-phase synthesis carrier, and finally A tetrapeptide fragment II) with a purity of 98-99% is obtained, and then the hexapeptide resin fragment III and the tetrapeptide fragment II are condensed to form a whole peptide resin, and finally degarelix acetate is obtained by cutting and salting. Among amino acids, NH 2 -Aph(Hor)-OH, which is prone to cause side reactions, is located at the sixth place. The present invention adopts a solid-phase synthesis 6+4 fragment synthesis method, which is equivalent to Aph(Hor) and alkali only once during the condensation process. Contact (after Aph(Hor) condensation only needs to be deprotected in an alkaline environment), compared to condensing amino acids one by one and repeatedly contacting with alkali multiple times, it effectively reduces the occurrence of side reactions. Moreover, the present invention uses the condensing agent MYMsA and/or MYTsA in the condensation process of the hexapeptide resin fragment III and the tetrapeptide fragment II. These two condensing agents have no racemization effect, strong condensation ability, and do not need to add any auxiliary Agents and catalysts help reduce the consumption of fragments. The above two reasons make the synthesis yield of degarelix acetate significantly improved, side reactions are significantly reduced, and the synthesis cost is correspondingly reduced.
具体地，采用本发明方法制备醋酸地加瑞克，氨基酸Aph(Hor)侧链重排杂质小于0.1％，粗肽纯度可达93～95％，提纯后纯度能够达到99.94％以上，总收率可达到80％以上。同时，该路线后处理操作简单，两个片段可以同时生产，然后组装成终产品，提高了生产放大的效率。能够应用于工业化生产。Specifically, using the method of the present invention to prepare degarelix acetate, the amino acid Aph (Hor) side chain rearrangement impurities is less than 0.1%, the purity of the crude peptide can reach 93-95%, and the purity after purification can reach more than 99.94%, and the total yield Can reach more than 80%. At the same time, the post-processing operation of this route is simple, and the two segments can be produced at the same time and then assembled into the final product, which improves the efficiency of production amplification. Can be applied to industrial production.
在一种优选的实施方式中，上述步骤S1包括：S11，将Rink Amide MBHA树脂依次进行溶胀、Fmoc脱保护后，将其与Fmoc-D-Ala-OH进行第一次缩合，得到第一缩合产物；S12，将第一缩合产物进行Fmoc脱保护后，将其与Fmoc-D-Ala-OH进行第二次缩合，得到第二缩合产物；S13，将第二缩合产物进行Fmoc脱保护后，将其与Fmoc-iLys(Boc)-OH进行第三次缩合，得到第三缩合产物；S14，将第三缩合产物进行Fmoc脱保护后，将其与Fmoc-Leu-OH 进行第四次缩合，得到第四缩合产物；S15，将第四缩合产物进行Fmoc脱保护后，将其与Fmoc-D-Aph(Cbm)-OH进行第五次缩合，得到第五缩合产物；S16，将第五缩合产物进行Fmoc脱保护后，将其与Fmoc-Aph(Hor)-OH进行第六次缩合，得到第六缩合产物，即为六肽树脂片段III。这样通过逐步的脱保护、缩合，将前六个氨基酸连接至Rink Amide MBHA树脂上，且Fmoc-Aph(Hor)-OH位于最末端，与碱进行了一次接触。In a preferred embodiment, the above step S1 includes: S11, after the Rink Amide MBHA resin is swollen and Fmoc deprotected in sequence, it is subjected to the first condensation with Fmoc-D-Ala-OH to obtain the first condensation Product; S12, after the first condensation product is Fmoc deprotected, it is condensed with Fmoc-D-Ala-OH for the second time to obtain the second condensation product; S13, after the second condensation product is Fmoc deprotected, Condense it with Fmoc-iLys(Boc)-OH for the third time to obtain the third condensation product; S14, after deprotecting the third condensation product by Fmoc, it is condensed with Fmoc-Leu-OH for the fourth time, Obtain the fourth condensation product; S15, after the fourth condensation product is Fmoc deprotected, it is condensed with Fmoc-D-Aph(Cbm)-OH for the fifth time to obtain the fifth condensation product; S16, the fifth condensation product is obtained After the product is Fmoc deprotected, it is condensed with Fmoc-Aph(Hor)-OH for the sixth time to obtain the sixth condensation product, which is the hexapeptide resin fragment III. In this way, through gradual deprotection and condensation, the first six amino acids are connected to Rink Amide MBHA resin, and Fmoc-Aph(Hor)-OH is located at the very end, making one contact with the base.
在一种优选的实施方式中，上述步骤S2包括：S21，将CTC树脂进行溶胀后(CTC树脂不用去保护，可以直接和氨基酸在碱性环境下反应，反应生成的一分子HCl与碱中和)，与Fmoc-Ser(tBu)-OH进行第七次缩合，得到第七缩合产物；S22，将第七缩合产物进行Fmoc脱保护后，将其与Fmoc-3-(3-pyridy)-D-Ala-OH进行第八次缩合，得到第八缩合产物；S23，将第八缩合产物进行Fmoc脱保护后，将其与Fmoc-D-Phe(4-Cl)-OH进行第九次缩合，得到第九缩合产物；S24，将第九缩合产物进行Fmoc脱保护后，将其与Fmoc-D-2Nal-OH进行第十次缩合，得到第十缩合产物，即为四肽树脂片段I。In a preferred embodiment, the above-mentioned step S2 includes: S21, after swelling the CTC resin (the CTC resin does not need to be deprotected, it can directly react with amino acids in an alkaline environment, and a molecule of HCl generated by the reaction is neutralized with alkali ), and Fmoc-Ser(tBu)-OH for the seventh condensation to obtain the seventh condensation product; S22, after the seventh condensation product is Fmoc deprotected, it is combined with Fmoc-3-(3-pyridy)-D -Ala-OH conducts the eighth condensation to obtain the eighth condensation product; S23, after the eighth condensation product is Fmoc deprotected, it is subjected to the ninth condensation with Fmoc-D-Phe(4-Cl)-OH, The ninth condensation product is obtained; S24, after the ninth condensation product is Fmoc deprotected, it is condensed with Fmoc-D-2Nal-OH for the tenth time to obtain the tenth condensation product, which is the tetrapeptide resin fragment I.
为了提高缩合效率，进一步减少副反应发生，在一种优选的实施方式中，第一次缩合至第六次缩合步骤中，以及第八次缩合至第十次缩合步骤中，采用的缩合体系分别独立地选自以下任一种：HOBT/DIC、HOAT/DIC、Oxymapure/DIC、HATU/DIPEA、HBTU/DIPEA、TBTU/DIPEA、PyBOP/DIPEA；第七次缩合步骤在碱性环境中进行，且采用的碱为DIPEA。(DIPEA只是提供碱性环境，捕获脱下来的HCl，该步骤中不需要缩合剂)。这里的缩合体系中，DIC是活化剂，HOBT、HOAT、Oxymapure是消旋抑制剂，DIPEA是碱，前面的HATU，HBTU，TBTU，PyBoP是活化剂。优选地，各Fmoc脱保护步骤中采用的脱保护试剂均为20wt％哌啶的DMF溶液。In order to improve the condensation efficiency and further reduce the occurrence of side reactions, in a preferred embodiment, the condensation systems used in the first condensation to the sixth condensation step and the eighth condensation to the tenth condensation step are respectively Independently selected from any of the following: HOBT/DIC, HOAT/DIC, Oxymapure/DIC, HATU/DIPEA, HBTU/DIPEA, TBTU/DIPEA, PyBOP/DIPEA; the seventh condensation step is carried out in an alkaline environment, and The base used is DIPEA. (DIPEA only provides an alkaline environment to capture the removed HCl, no condensation agent is needed in this step). In the condensation system here, DIC is the activator, HOBT, HOAT, and Oxymapure are racemization inhibitors, DIPEA is the base, and HATU, HBTU, TBTU, and PyBoP are the activators. Preferably, the deprotection reagent used in each Fmoc deprotection step is a DMF solution of 20 wt% piperidine.
在一种优选的实施方式中，上述步骤S1和步骤S2中，各缩合反应过程中采用的反应溶剂为DMF、DCM、THF中的一种或多种。上述几种溶剂对氨基酸有较好的溶解性，能够提供更稳定的缩合反应环境。且为了进一步提高各氨基酸缩合反应的效率，在一种优选的实施方式中，上述步骤S1和步骤S2中，各缩合反应过程中的反应温度为10～35℃，反应时间为0.5～4h。In a preferred embodiment, in the above steps S1 and S2, the reaction solvent used in each condensation reaction process is one or more of DMF, DCM, and THF. The above-mentioned solvents have good solubility for amino acids and can provide a more stable environment for the condensation reaction. And in order to further improve the efficiency of the condensation reaction of each amino acid, in a preferred embodiment, in the above step S1 and step S2, the reaction temperature during each condensation reaction process is 10 to 35° C., and the reaction time is 0.5 to 4 h.
在一种优选的实施方式中，上述步骤S1和步骤S2中，各缩合反应过程中，保护氨基酸、缩合体系及Fmoc氨基树脂之间的摩尔比为1.5～3:1.5～6:1，其中Fmoc氨基树脂的摩尔数以所含Fmoc保护氨基的摩尔数计。这里的保护氨基酸指的是每次氨基酸缩合反应过程中的Fmoc保护氨基酸，比如第一次缩合步骤中的Fmoc-D-Ala-OH，第二次缩合步骤中的Fmoc-D-Ala-OH。Fmoc氨基树脂指的是各缩合反应过程中的树脂一方，比如第一次缩合步骤中的Rink Amide MBHA树脂，第二次缩合步骤中的第一缩合产物，且Fmoc氨基树脂的摩尔数以所含Fmoc保护氨基的摩尔数计，相当于按照能够进行反应的位点数目计。将保护氨基酸、缩合体系及Fmoc氨基树脂之间的摩尔比控制在上述范围，既有利于提高反应效率、转化率，也能够减少原料浪费。In a preferred embodiment, in the above step S1 and step S2, during each condensation reaction process, the molar ratio between the protected amino acid, the condensation system and the Fmoc amino resin is 1.5 to 3: 1.5 to 6:1, where Fmoc The number of moles of amino resin is based on the number of moles of Fmoc protected amino group contained. The protected amino acid here refers to the Fmoc protected amino acid during each amino acid condensation reaction, such as Fmoc-D-Ala-OH in the first condensation step, and Fmoc-D-Ala-OH in the second condensation step. Fmoc amino resin refers to the resin side in each condensation reaction process, such as Rink Amide MBHA resin in the first condensation step, the first condensation product in the second condensation step, and the number of moles of Fmoc amino resin is The number of moles of Fmoc protected amino groups is equivalent to the number of sites that can be reacted. Controlling the molar ratio between the protected amino acid, the condensation system and the Fmoc amino resin within the above range not only helps to improve the reaction efficiency and conversion rate, but also reduces the waste of raw materials.
为了进一步促进缩合反应的进行，提高反应效率和各缩合反应过程中的产物收率，在一种优选的实施方式中，Rink Amide MBHA树脂的取代度为0.6～1.0mmol/g，CTC树脂的取代度为0.9～1.6mmol/g。In order to further promote the progress of the condensation reaction and improve the reaction efficiency and the product yield in each condensation reaction process, in a preferred embodiment, the degree of substitution of Rink Amide MBHA resin is 0.6-1.0 mmol/g, and the substitution of CTC resin The degree is 0.9～1.6mmol/g.
在一种优选的实施方式中，在切割四肽树脂片段I的树脂的步骤之前，步骤S3还包括对四肽树脂片段I依次进行Fmoc脱保护、封端反应的步骤，具体如下：采用20wt％哌啶的DMF溶液对四肽树脂片段I进行Fmoc脱保护，得到脱保护片段I；将脱保护片段I与乙酸酐、DIPEA的作用下进行封端反应，得到封端后的四肽树脂片段I。这样可以将四肽树脂片段I中未参与缩合反应的氨基封端，避免其参与后续反应，从而可以进一步减少副反应的发生。In a preferred embodiment, before the step of cutting the resin of the tetrapeptide resin fragment I, step S3 further includes the steps of sequentially performing Fmoc deprotection and end-capping reactions on the tetrapeptide resin fragment I, specifically as follows: 20wt% The DMF solution of piperidine performs Fmoc deprotection on the tetrapeptide resin fragment I to obtain the deprotected fragment I; the deprotected fragment I is subjected to a capping reaction under the action of acetic anhydride and DIPEA to obtain the capped tetrapeptide resin fragment I . In this way, the amino groups in the tetrapeptide resin fragment I that are not involved in the condensation reaction can be capped to prevent them from participating in subsequent reactions, thereby further reducing the occurrence of side reactions.
在一种优选的实施方式中，上述步骤S4包括：S41，将六肽树脂片段III进行Fmoc脱保护，得到脱保护片段III；S42，将四肽片段II和缩合剂、反应溶剂混合，得到预处理液；S43，将预处理液与脱保护片段III进行缩合反应，得到全肽树脂。Fmoc脱保护后，六肽树脂片段III中末端氨基露出，然后与四肽片段II的末端羟基发生缩合反应，该过程中，六肽树脂片段III的末端Aph(Hor)第二次与碱接触，反应后即可形成全肽树脂，极大地降低了副反应发生的几率。In a preferred embodiment, the above step S4 includes: S41, Fmoc deprotection of the hexapeptide resin fragment III to obtain the deprotected fragment III; S42, mixing the tetrapeptide fragment II with the condensing agent and the reaction solvent to obtain the pre Treatment solution; S43, the pretreatment solution and the deprotected fragment III are subjected to a condensation reaction to obtain a whole peptide resin. After Fmoc is deprotected, the terminal amino group of the hexapeptide resin fragment III is exposed, and then it undergoes a condensation reaction with the terminal hydroxyl group of the tetrapeptide fragment II. During this process, the terminal Aph(Hor) of the hexapeptide resin fragment III is in contact with alkali for the second time. After the reaction, the whole peptide resin can be formed, which greatly reduces the probability of side reactions.
为了提供更稳定的反应环境，同时提高反应效率，在一种优选的实施方式中，上述步骤S42中采用的反应溶剂为DMF、DCM、THF中的一种或多种；相对于六肽树脂片段III，四肽片段II的当量为1.2～1.5eq，缩合剂MYMsA和/或MYTsA的当量为1.2～1.5eq。更优选地，在得到全肽树脂后，步骤S43还包括对全肽树脂进行洗涤、收缩、氮气吹扫烘干的步骤。In order to provide a more stable reaction environment and improve reaction efficiency, in a preferred embodiment, the reaction solvent used in step S42 is one or more of DMF, DCM, and THF; compared to the hexapeptide resin fragment III, the equivalent of the tetrapeptide fragment II is 1.2-1.5 eq, and the equivalent of the condensing agent MYMsA and/or MYTsA is 1.2-1.5 eq. More preferably, after the whole peptide resin is obtained, step S43 further includes the steps of washing, shrinking, and nitrogen purging and drying the whole peptide resin.
在一种优选的实施方式中，上述步骤S5包括：采用切割液与全肽树脂反应后，过滤，得到滤液；其中切割液为95vol％TFA的水溶液；采用甲基叔丁基醚对滤液逼晶，过滤，得到地加瑞克的粗产物；对粗产物进行提纯，得到地加瑞克。采用上述切割工序，能够将全肽树脂中的线性肽链完整分离。且为了进一步提高提纯效果，优选地，上述提纯过程采用反向高效液相色谱法，具体采用AQ填料，A相采用0.05wt％TFA的水溶液，B相采用0.05wt％TFA的乙腈溶液。更优选地，上述步骤S6包括：采用醋酸水溶液对地加瑞克进行转盐，得到醋酸地加瑞克。具体地，可以在反向高效液相色谱法提纯后，以1wt％AcOH的水溶液与乙腈作为流动相梯度洗脱并转盐，后冻干，得到醋酸地加瑞克产品。In a preferred embodiment, the above step S5 includes: after the cleavage fluid is reacted with the whole peptide resin, filtered to obtain the filtrate; wherein the cleavage fluid is a 95 vol% TFA aqueous solution; and methyl tert-butyl ether is used to force the filtrate to crystallize , Filter to obtain the crude product of degarelix; purify the crude product to obtain degarelix. Using the above cutting process, the linear peptide chains in the whole peptide resin can be completely separated. And in order to further improve the purification effect, preferably, the above purification process adopts reverse high performance liquid chromatography, specifically using AQ packing, phase A adopts 0.05wt% TFA aqueous solution, and phase B adopts 0.05wt% TFA acetonitrile solution. More preferably, the above step S6 includes: using an aqueous solution of acetic acid to salt degarelix to obtain degarelix acetate. Specifically, after purification by reverse-phase high performance liquid chromatography, a 1 wt% AcOH aqueous solution and acetonitrile are used as mobile phase gradient elution and salt conversion, and then freeze-dried to obtain the degarelix acetate product.
上述缩写代表的试剂如下：The reagents represented by the above abbreviations are as follows:
Figure PCTCN2019100530-appb-000003
Figure PCTCN2019100530-appb-000003
Figure PCTCN2019100530-appb-000004
Figure PCTCN2019100530-appb-000004
以下结合具体实施例对本申请作进一步详细描述，这些实施例不能理解为限制本申请所要求保护的范围。The application will be further described in detail below in conjunction with specific embodiments, and these embodiments should not be construed as limiting the scope of protection claimed by the application.
实施例1Example 1
该实施例1中合成了醋酸地加瑞克，具体工艺如下：In Example 1, degarelix acetate was synthesized, and the specific process is as follows:
六肽树脂片段III的合成：Synthesis of hexapeptide resin fragment III:
反应柱中依次加入取代度为0.68mmol/g的Reink amide MBHA树脂100g，使用DMF溶胀30min后抽滤除去溶剂，然后在反应柱中加入20wt％哌啶的DMF溶液进行Fmoc脱保护，反应30min后滤除溶液，树脂使用DMF洗涤六遍。Add 100g of Reinkamide MBHA resin with a degree of substitution of 0.68mmol/g to the reaction column, swell with DMF for 30 minutes, and then remove the solvent by suction filtration. Then add 20wt% piperidine in DMF solution to the reaction column for Fmoc deprotection. After 30 minutes of reaction The solution was filtered off, and the resin was washed six times with DMF.
四口瓶中加入42.3g Fmoc-D-Ala-OH(136mmol)，19.3g Oxymapure(136mmol)，控温0～5℃加入17.2g DIC(136mmol)活化氨基酸5～10min。活化后的溶液转移至多肽反应釜中用氮气鼓泡反应，控温20～25℃反应2h后使用茚三酮显色法进行反应跟踪，树脂无色透明则反应完全，树脂颗粒显色则未反应完全，需要延长反应直至树脂无色透明。重复上述脱保护步骤及缩合步骤按照肽链顺序分别缩合连接Fmoc-Pro-OH，Fmoc-iLys(Boc)-OH，Fmoc-Leu-OH，Fmoc-D-Aph(Cbm)-OH，Fmoc-Aph(Hor)-OH，得到六肽树脂片段III。Add 42.3g of Fmoc-D-Ala-OH (136mmol), 19.3g of Oxymapure (136mmol) into a four-necked flask, add 17.2g of DIC (136mmol) to activate the amino acid for 5-10min at 0～5℃. The activated solution is transferred to the peptide reactor and reacted by bubbling with nitrogen. The temperature is controlled at 20～25℃ and the reaction is carried out for 2h. The reaction is followed by the ninhydrin color method. If the resin is colorless and transparent, the reaction is complete, and the resin particles are not colored. The reaction is complete and the reaction needs to be extended until the resin is colorless and transparent. Repeat the above-mentioned deprotection and condensation steps to condense and connect Fmoc-Pro-OH, Fmoc-iLys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Aph(Cbm)-OH, Fmoc-Aph according to the sequence of the peptide chain. (Hor)-OH to obtain hexapeptide resin fragment III.
四肽片段II的合成：Synthesis of tetrapeptide fragment II:
反应柱中依次加入取代度为1.15mmol/g的CTC树脂85g，使用DMF溶胀30min后抽滤除去溶剂，四口瓶加入112.4g Fmoc-Ser(tBu)-OH(293.2mmol)，DMF 850ml溶解氨基酸，加入DIPEA 75.8g(586.4mmol)搅拌10min后体系转移至多肽反应柱中用氮气鼓泡反应2h后滤除反应液。DMF洗涤树脂6次，然后反应柱依次加入DMF，甲醇(85ml)，DIPEA 75.8g(586.4mmol)。控温20～25℃反应2h以对剩余的活性位点进行封端，反完毕后DMF洗涤树脂6遍，甲醇二氯甲烷交替洗涤收缩树脂。得到Fmoc-Ser(tBu)-CTC resin树脂120g，取代度0.80mmol/g。Add 85 g of CTC resin with a degree of substitution of 1.15 mmol/g to the reaction column, swell with DMF for 30 minutes, and then remove the solvent by suction filtration. Add 112.4 g Fmoc-Ser(tBu)-OH (293.2 mmol) in a four-neck bottle, and DMF 850 ml to dissolve amino acids After adding DIPEA 75.8g (586.4mmol) and stirring for 10 minutes, the system was transferred to the peptide reaction column and reacted with nitrogen for 2 hours, and then the reaction solution was filtered out. DMF washes the resin 6 times, and then adds DMF, methanol (85ml) and DIPEA 75.8g (586.4mmol) to the reaction column. The temperature was controlled at 20-25°C to react for 2 hours to cap the remaining active sites. After the reaction, the resin was washed with DMF 6 times, and the shrink resin was washed alternately with methanol and dichloromethane. Obtain 120 g of Fmoc-Ser(tBu)-CTC resin with a degree of substitution of 0.80 mmol/g.
称取120g取代度为0.80mmol/g的Fmoc-Ser(tBu)-CTC resin树脂(96mmol)加入固相反应器中，用DMF溶胀30min后抽滤除去DMF，然后反应柱中加入20wt％哌啶的DMF溶液进行Fmoc脱保护，反应30min后滤除溶液，树脂使用DMF洗涤六遍。Weigh 120g of Fmoc-Ser(tBu)-CTC resin (96mmol) with a degree of substitution of 0.80mmol/g into the solid phase reactor, swell with DMF for 30 minutes and remove DMF by suction, then add 20wt% piperidine to the reaction column Fmoc deprotection was performed on the DMF solution of the polymer, the solution was filtered off after 30 minutes of reaction, and the resin was washed with DMF six times.
称取Fmoc-3-(3-pyridy)-D-Ala-OH(74.6g,192.1mmol)，Oxymapure(27.3g,192.1mmol)加入DMF溶解，控温0～5℃加入DIC(24.2g,191.6mmol)，加毕搅拌5～10min后转移至反应釜进行反应。反应2h后使用茚三酮显色法进行反应跟踪，直至树脂无色透明则反应完全。重复上述脱保护步骤及缩合步骤按照肽链顺序分别缩合连接Fmoc-D-Phe(4-Cl)-OH和Fmoc-D-2Nal-OH。Weigh Fmoc-3-(3-pyridy)-D-Ala-OH (74.6g, 192.1mmol), add Oxymapure (27.3g, 192.1mmol) to DMF to dissolve, control the temperature at 0～5℃ and add DIC (24.2g, 191.6 mmol), after addition, stirring for 5-10 min, then transfer to the reactor for reaction. After reaction for 2 hours, the reaction was followed by the ninhydrin color method until the resin was colorless and transparent. Repeat the above-mentioned deprotection step and condensation step to condense and connect Fmoc-D-Phe(4-Cl)-OH and Fmoc-D-2Nal-OH respectively according to the sequence of the peptide chain.
连接完肽链后，继续加入20wt％哌啶的DMF溶液进行Fmoc脱保护，反应30min后滤除溶液，树脂使用DMF洗涤六遍。其次，加入乙酸酐/DIPEA＝3：6(eq)反应1h进行封端反应。反应完，用甲醇/二氯甲烷收缩树脂并干燥，得到四肽树脂片段I。After connecting the peptide chain, continue to add 20wt% piperidine in DMF solution for Fmoc deprotection. After 30 minutes of reaction, the solution was filtered out, and the resin was washed with DMF six times. Secondly, add acetic anhydride/DIPEA=3:6 (eq) and react for 1 h to perform end-capping reaction. After the reaction, the resin was shrunk with methanol/dichloromethane and dried to obtain the tetrapeptide resin fragment I.
使用0.5wt％TFA/DCM溶液对四肽树脂片段I进行切割反应2h，滤液浓缩后使用甲基叔丁基醚逼晶，过滤，得到四肽片段II，共计66.6g，收率95％。The tetrapeptide resin fragment I was cleaved with 0.5wt% TFA/DCM solution for 2h. After the filtrate was concentrated, methyl tert-butyl ether was used to crystallize and filtered to obtain tetrapeptide fragment II, totaling 66.6 g, with a yield of 95%.
全肽树脂的合成：Synthesis of whole peptide resin:
向反应柱的六肽树脂片段III中加入20wt％哌啶的DMF溶液进行Fmoc脱保护，反应30min后滤除溶液，树脂使用DMF洗涤六遍。Add 20wt% piperidine in DMF solution to the hexapeptide resin fragment III of the reaction column for Fmoc deprotection. After 30 minutes of reaction, the solution was filtered out, and the resin was washed with DMF six times.
称取四肽片段II(59.6g，1.2eq)，加入DMF溶解，控温20～25℃加入MYMsA(10.85g，1.2eq)活化四肽片段II形成活性中间体，加毕搅拌2～3h后转移至反应柱进行反应。反应2h后使用茚三酮显色法进行反应跟踪，树脂有颜色则继续反应。无色透明则反应完全。反应完毕后抽去溶剂，树脂使用DMF洗涤6遍。Weigh the tetrapeptide fragment II (59.6g, 1.2eq), add DMF to dissolve it, add MYMsA (10.85g, 1.2eq) to activate the tetrapeptide fragment II to form an active intermediate at 20～25℃, and stir for 2～3h after adding Transfer to the reaction column for reaction. After reacting for 2 hours, the reaction was followed by the ninhydrin color method, and the reaction continued when the resin was colored. Colorless and transparent, the reaction is complete. After the reaction was completed, the solvent was removed, and the resin was washed 6 times with DMF.
树脂使用甲醇与二氯甲烷交替洗涤收缩，收缩完毕氮气吹扫烘料至恒重后收料，得到全肽树脂。The resin is washed and shrunk alternately with methanol and dichloromethane. After the shrinkage is completed, the nitrogen purge and bake the material to a constant weight and then the material is collected to obtain the peptide resin.
地加瑞克粗产物的制备：Preparation of degarelix crude product:
连接完毕的全肽树脂使用95vol％TFA/5vol％H 2O比例切割液反应3～5h，滤液浓缩后使用甲基叔丁基醚逼晶，过滤，得到终产品104.3g，粗肽收率94％，纯度95％。 The connected whole peptide resin is reacted with 95vol%TFA/5vol%H 2 O ratio cutting solution for 3～5h. After the filtrate is concentrated, methyl tert-butyl ether is used to crystallize and filter to obtain 104.3g of the final product. The yield of crude peptide is 94 %, 95% purity.
纯化、转盐：Purification and salt transfer:
采用反向液相色谱法纯化：用AQ的填料，A相：0.05wt％TFA的水溶液；B相：0.05wt％TFA的乙腈溶液制备纯化，转盐使用1wt％AcOH的水溶液与乙腈作为流动相梯度洗脱后冻干，得到最终产品醋酸地加瑞克。HPLC测纯度，见图1，纯度为99.94％，最大单杂含量为0.06％，＜0.1％。质谱测量结果见图2，目标产物分子量M＝1632.3，下图中1633.8＝M+1，816.9＝(M/2)+1，545.4＝(M/3)+1，换算为醋酸地加瑞克的总收率为80.8％。Purification by reverse liquid chromatography: use AQ packing, phase A: 0.05wt% TFA aqueous solution; Phase B: 0.05wt% TFA acetonitrile solution for preparation and purification, and use 1wt% AcOH aqueous solution and acetonitrile as mobile phase for salt conversion After gradient elution, it was lyophilized to obtain the final product degarelix acetate. The purity measured by HPLC is shown in Figure 1. The purity is 99.94%, and the maximum single impurity content is 0.06%, <0.1%. The results of mass spectrometry are shown in Figure 2. The molecular weight of the target product is M = 1632.3. In the figure below, 1633.8 = M+1, 816.9 = (M/2) + 1, 545.4 = (M/3) + 1, converted to degarelix acetate The total yield was 80.8%.
实施例2Example 2
与实施例1的区别为：各缩合反应步骤中，保护氨基酸、相应的缩合体系及Fmoc氨基树脂的物质量之比为3:1.5:1，且缩合体系中各成分比例不变。产品的收率为86％，纯度为82％。The difference from Example 1 is that in each condensation reaction step, the ratio of the amount of the protected amino acid, the corresponding condensation system and the Fmoc amino resin is 3:1.5:1, and the ratio of each component in the condensation system remains unchanged. The yield of the product was 86%, and the purity was 82%.
实施例3Example 3
与实施例1的区别为：各缩合反应步骤中，保护氨基酸、相应的缩合体系及Fmoc氨基树脂的物质量之比为1.5:3:1，且缩合体系中各成分比例不变。产品的收率为87％，纯度为83％。The difference from Example 1 is that in each condensation reaction step, the ratio of the amount of the protected amino acid, the corresponding condensation system and the Fmoc amino resin is 1.5:3:1, and the ratio of each component in the condensation system remains unchanged. The yield of the product was 87%, and the purity was 83%.
实施例4Example 4
与实施例1的区别为：各缩合反应步骤中，保护氨基酸、相应的缩合体系及Fmoc氨基树脂的物质量之比为1.2:1.2:1，且缩合体系中各成分比例不变。产品的收率为85％，纯度为79％。The difference from Example 1 is that in each condensation reaction step, the ratio of the amount of the protected amino acid, the corresponding condensation system and the Fmoc amino resin is 1.2:1.2:1, and the ratio of each component in the condensation system remains unchanged. The yield of the product is 85% and the purity is 79%.
实施例5Example 5
与实施例1的区别为：六肽树脂片段III和四肽树脂片段II合成过程的各缩合反应步骤中，相应的缩合体系为DIC/HOBT，其他成份比列不变，粗肽收率为93％，纯度为94％。The difference from Example 1 is that in each condensation reaction step of the synthesis process of hexapeptide resin fragment III and tetrapeptide resin fragment II, the corresponding condensation system is DIC/HOBT, and the ratio of other components remains unchanged. The yield of crude peptide is 93 %, the purity is 94%.
实施例6Example 6
与实施例1的区别为：六肽树脂片段III和四肽树脂片段II合成过程的各缩合反应步骤中，相应的缩合体系为HBTU/DIPEA，其他成份比列不变，粗肽收率为92％，纯度为95％。The difference from Example 1 is that in each condensation reaction step of the synthesis process of hexapeptide resin fragment III and tetrapeptide resin fragment II, the corresponding condensation system is HBTU/DIPEA, and the ratio of other components remains unchanged. The yield of crude peptide is 92 %, the purity is 95%.
实施例7Example 7
与实施例1的区别为：六肽树脂片段III和四肽树脂片段II合成过程的各缩合反应步骤中，相应的缩合体系为PyBoP/DIPEA，其他成份比列不变，粗肽收率为95％，纯度为93％。The difference from Example 1 is that in each condensation reaction step of the synthesis process of hexapeptide resin fragment III and tetrapeptide resin fragment II, the corresponding condensation system is PyBoP/DIPEA, and the ratio of other components remains unchanged. The yield of crude peptide is 95 %, the purity is 93%.
对比例1Comparative example 1
与实施例1的区别为：全肽树脂缩合体系为HATU/DIPEA，粗肽收率为75％，纯度为81％。The difference from Example 1 is: the whole peptide resin condensation system is HATU/DIPEA, the crude peptide yield is 75%, and the purity is 81%.
对比例2Comparative example 2
与实施例1的区别为：全肽树脂缩合体系为PyBOP/DIPEA。粗肽收率为77％，纯度为79％。The difference from Example 1 is: the whole peptide resin condensation system is PyBOP/DIPEA. The yield of the crude peptide was 77% and the purity was 79%.
对比例3Comparative example 3
与实施例1的区别为：全肽树脂缩合体系为DIC/HOBT。粗肽收率为86％，纯度为82％。The difference from Example 1 is that the whole peptide resin condensation system is DIC/HOBT. The yield of the crude peptide was 86% and the purity was 82%.
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The foregoing descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc., made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (14)

  1. 一种醋酸地加瑞克的合成方法，其特征在于，包括：A method for synthesizing degarelix acetate, which is characterized in that it comprises:
    S1，以Rink Amide MBHA树脂为固相合成的载体，将其按照氨基酸顺序依次与以下六种保护氨基酸Fmoc-D-Ala-OH、Fmoc-Pro-OH、Fmoc-iLys(Boc)-OH、Fmoc-Leu-OH、Fmoc-D-Aph(Cbm)-OH及Fmoc-Aph(Hor)-OH进行缩合反应，得到六肽树脂片段III；S1, using Rink Amide MBHA resin as the carrier for solid-phase synthesis, and combining it with the following six protected amino acids Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-iLys(Boc)-OH, Fmoc in the sequence of amino acids -Leu-OH, Fmoc-D-Aph(Cbm)-OH and Fmoc-Aph(Hor)-OH undergo condensation reaction to obtain hexapeptide resin fragment III;
    S2，以CTC树脂为固相合成的载体，将其按照氨基酸顺序依次与以下四种保护氨基酸Fmoc-Ser(tBu)-OH、Fmoc-3-(3-pyridy)-D-Ala-OH、Fmoc-D-Phe(4-Cl)-OH及Fmoc-D-2Nal-OH进行缩合反应，得到四肽树脂片段I；S2, CTC resin is used as a solid-phase synthesis carrier, and the following four protective amino acids Fmoc-Ser(tBu)-OH, Fmoc-3-(3-pyridy)-D-Ala-OH, Fmoc -D-Phe(4-Cl)-OH and Fmoc-D-2Nal-OH undergo condensation reaction to obtain tetrapeptide resin fragment I;
    S3，切割所述四肽树脂片段I的树脂，得到四肽片段II；S3, cutting the resin of the tetrapeptide resin fragment I to obtain the tetrapeptide fragment II;
    S4，将所述六肽树脂片段III与所述四肽片段II在缩合剂MYMsA和/或MYTsA的作用下进行缩合反应，得到全肽树脂；S4, the hexapeptide resin fragment III and the tetrapeptide fragment II are subjected to a condensation reaction under the action of a condensation agent MYMsA and/or MYTsA to obtain a whole peptide resin;
    S5，切割所述全肽树脂的树脂，得到地加瑞克；S5, cutting the resin of the whole peptide resin to obtain degarelix;
    S6，对所述地加瑞克进行醋酸转盐，得到所述醋酸地加瑞克。S6: Acetate the degarelix to obtain the degarelix acetate.
  2. 根据权利要求1所述的合成方法，其特征在于，所述步骤S1包括：The synthesis method according to claim 1, wherein the step S1 comprises:
    S11，将所述Rink Amide MBHA树脂依次进行溶胀、Fmoc脱保护后，将其与所述Fmoc-D-Ala-OH进行第一次缩合，得到第一缩合产物；S11, after the Rink Amide MBHA resin is swollen and Fmoc deprotected in sequence, it is condensed with the Fmoc-D-Ala-OH for the first time to obtain a first condensation product;
    S12，将所述第一缩合产物进行Fmoc脱保护后，将其与所述Fmoc-D-Ala-OH进行第二次缩合，得到第二缩合产物；S12, after the first condensation product is subjected to Fmoc deprotection, it is subjected to a second condensation with the Fmoc-D-Ala-OH to obtain a second condensation product;
    S13，将所述第二缩合产物进行Fmoc脱保护后，将其与所述Fmoc-iLys(Boc)-OH进行第三次缩合，得到第三缩合产物；S13, after the second condensation product is subjected to Fmoc deprotection, it is subjected to a third condensation with the Fmoc-iLys(Boc)-OH to obtain a third condensation product;
    S14，将所述第三缩合产物进行Fmoc脱保护后，将其与所述Fmoc-Leu-OH进行第四次缩合，得到第四缩合产物；S14, after the third condensation product is subjected to Fmoc deprotection, it is subjected to a fourth condensation with the Fmoc-Leu-OH to obtain a fourth condensation product;
    S15，将所述第四缩合产物进行Fmoc脱保护后，将其与所述Fmoc-D-Aph(Cbm)-OH进行第五次缩合，得到第五缩合产物；S15: After the fourth condensation product is subjected to Fmoc deprotection, it is subjected to a fifth condensation with the Fmoc-D-Aph(Cbm)-OH to obtain a fifth condensation product;
    S16，将所述第五缩合产物进行Fmoc脱保护后，将其与所述Fmoc-Aph(Hor)-OH进行第六次缩合，得到第六缩合产物，即为所述六肽树脂片段III。S16: After the fifth condensation product is Fmoc deprotected, it is condensed with the Fmoc-Aph(Hor)-OH for the sixth time to obtain a sixth condensation product, which is the hexapeptide resin fragment III.
  3. 根据权利要求2所述的合成方法，其特征在于，所述步骤S2包括：The synthesis method according to claim 2, wherein the step S2 comprises:
    S21，将所述CTC树脂进行溶胀后，与所述Fmoc-Ser(tBu)-OH进行第七次缩合，得到第七缩合产物；S21, after swelling the CTC resin, it is condensed with the Fmoc-Ser(tBu)-OH for the seventh time to obtain a seventh condensation product;
    S22，将所述第七缩合产物进行Fmoc脱保护后，将其与所述 Fmoc-3-(3-pyridy)-D-Ala-OH进行第八次缩合，得到第八缩合产物；S22, after the seventh condensation product is subjected to Fmoc deprotection, the seventh condensation product is subjected to the eighth condensation with the Fmoc-3-(3-pyridy)-D-Ala-OH to obtain an eighth condensation product;
    S23，将所述第八缩合产物进行Fmoc脱保护后，将其与所述Fmoc-D-Phe(4-Cl)-OH进行第九次缩合，得到第九缩合产物；S23, after the eighth condensation product is subjected to Fmoc deprotection, it is subjected to a ninth condensation with the Fmoc-D-Phe(4-Cl)-OH to obtain a ninth condensation product;
    S24，将所述第九缩合产物进行Fmoc脱保护后，将其与所述Fmoc-D-2Nal-OH进行第十次缩合，得到第十缩合产物，即为所述四肽树脂片段I。S24, after the ninth condensation product is subjected to Fmoc deprotection, it is subjected to a tenth condensation with the Fmoc-D-2Nal-OH to obtain a tenth condensation product, which is the tetrapeptide resin fragment I.
  4. 根据权利要求3所述的合成方法，其特征在于，所述第一次缩合至所述第六次缩合步骤中，以及所述第八次缩合至所述第十次缩合步骤中，采用的缩合体系分别独立地选自以下任一种：HOBT/DIC、HOAT/DIC、Oxymapure/DIC、HATU/DIPEA、HBTU/DIPEA、TBTU/DIPEA、PyBOP/DIPEA；所述第七次缩合步骤在碱性环境中进行，且采用的碱为DIPEA；The synthesis method according to claim 3, wherein the first condensation to the sixth condensation step, and the eighth condensation to the tenth condensation step, the condensation The system is independently selected from any of the following: HOBT/DIC, HOAT/DIC, Oxymapure/DIC, HATU/DIPEA, HBTU/DIPEA, TBTU/DIPEA, PyBOP/DIPEA; the seventh condensation step is in an alkaline environment The base used is DIPEA;
    优选地，各Fmoc脱保护步骤中采用的脱保护试剂均为20wt％哌啶的DMF溶液。Preferably, the deprotection reagent used in each Fmoc deprotection step is a DMF solution of 20 wt% piperidine.
  5. 根据权利要求1至4中任一项所述的合成方法，其特征在于，所述步骤S1和所述步骤S2中，各缩合反应过程中采用的反应溶剂为DMF、DCM、THF中的一种或多种。The synthesis method according to any one of claims 1 to 4, wherein in the step S1 and the step S2, the reaction solvent used in each condensation reaction process is one of DMF, DCM, and THF Or multiple.
  6. 根据权利要求5所述的合成方法，其特征在于，所述步骤S1和所述步骤S2中，各缩合反应过程中的反应温度为10～35℃，反应时间为0.5～4h。The synthesis method according to claim 5, characterized in that, in the step S1 and the step S2, the reaction temperature in each condensation reaction process is 10 to 35° C., and the reaction time is 0.5 to 4 h.
  7. 根据权利要求5所述的合成方法，其特征在于，所述步骤S1和所述步骤S2中，各缩合反应过程中，保护氨基酸、缩合体系及Fmoc氨基树脂之间的摩尔比为1.5～3:1.5～6:1，其中所述Fmoc氨基树脂的摩尔数以所含Fmoc保护氨基的摩尔数计。The synthesis method according to claim 5, wherein in the step S1 and the step S2, during each condensation reaction process, the molar ratio between the protected amino acid, the condensation system and the Fmoc amino resin is 1.5 to 3: 1.5-6:1, wherein the number of moles of the Fmoc amino resin is based on the number of moles of the Fmoc protected amino group.
  8. 根据权利要求1至4中任一项所述的合成方法，其特征在于，所述Rink Amide MBHA树脂的取代度为0.6～1.0mmol/g，所述CTC树脂的取代度为0.9～1.6mmol/g。The synthesis method according to any one of claims 1 to 4, wherein the degree of substitution of the Rink Amide MBHA resin is 0.6 to 1.0 mmol/g, and the degree of substitution of the CTC resin is 0.9 to 1.6 mmol/g. g.
  9. 根据权利要求1至4中任一项所述的合成方法，其特征在于，在切割所述四肽树脂片段I的树脂的步骤之前，所述步骤S3还包括对所述四肽树脂片段I依次进行Fmoc脱保护、封端反应的步骤，具体如下：The synthesis method according to any one of claims 1 to 4, characterized in that, before the step of cleaving the resin of the tetrapeptide resin fragment I, the step S3 further comprises sequentially analyzing the tetrapeptide resin fragment I The steps of Fmoc deprotection and capping reaction are as follows:
    采用20wt％哌啶的DMF溶液对所述四肽树脂片段I进行Fmoc脱保护，得到脱保护片段I；Fmoc deprotection of the tetrapeptide resin fragment I with 20wt% piperidine in DMF solution to obtain deprotected fragment I;
    将所述脱保护片段I与乙酸酐、DIPEA的作用下进行封端反应，得到封端后的所述四肽树脂片段I。The deprotected fragment I is subjected to a capping reaction under the action of acetic anhydride and DIPEA to obtain the capped tetrapeptide resin fragment I.
  10. 根据权利要求1至4中任一项所述的合成方法，其特征在于，所述步骤S4包括：The synthesis method according to any one of claims 1 to 4, wherein the step S4 comprises:
    S41，将所述六肽树脂片段III进行Fmoc脱保护，得到脱保护片段III；S41, Fmoc deprotection of the hexapeptide resin fragment III to obtain a deprotected fragment III;
    S42，将所述四肽片段II和所述缩合剂、反应溶剂混合，得到预处理液；S42, mixing the tetrapeptide fragment II with the condensing agent and the reaction solvent to obtain a pretreatment liquid;
    S43，将所述预处理液与所述脱保护片段III进行缩合反应，得到所述全肽树脂。S43: Perform a condensation reaction between the pretreatment liquid and the deprotected fragment III to obtain the whole peptide resin.
  11. 根据权利要求10所述的合成方法，其特征在于，所述步骤S42中采用的反应溶剂为DMF、DCM、THF中的一种或多种；相对于所述六肽树脂片段III，所述四肽片段II的当量为1.2～1.5eq，所述缩合剂MYMsA和/或MYTsA的当量为1.2～1.5eq。The synthesis method according to claim 10, wherein the reaction solvent used in the step S42 is one or more of DMF, DCM, and THF; compared to the hexapeptide resin fragment III, the four The equivalent of peptide fragment II is 1.2-1.5 eq, and the equivalent of the condensation agent MYMsA and/or MYTsA is 1.2-1.5 eq.
  12. 根据权利要求10所述的合成方法，其特征在于，在得到所述全肽树脂后，所述步骤S43还包括对所述全肽树脂进行洗涤、收缩、氮气吹扫烘干的步骤。The synthesis method according to claim 10, characterized in that, after the whole peptide resin is obtained, the step S43 further comprises the steps of washing, shrinking, and nitrogen purging and drying the whole peptide resin.
  13. 根据权利要求1至4中任一项所述的合成方法，其特征在于，所述步骤S5包括：The synthesis method according to any one of claims 1 to 4, wherein the step S5 comprises:
    采用切割液与所述全肽树脂反应后，过滤，得到滤液；其中所述切割液为95vol％TFA的水溶液；After reacting the cleavage fluid with the whole peptide resin, filter to obtain a filtrate; wherein the cleavage fluid is a 95 vol% TFA aqueous solution;
    采用甲基叔丁基醚对所述滤液逼晶，过滤，得到所述地加瑞克的粗产物；Using methyl tert-butyl ether to force the filtrate to crystallize and filter to obtain the crude product of degarelix;
    对所述粗产物进行提纯，得到所述地加瑞克。The crude product is purified to obtain the degarelix.
  14. 根据权利要求1至4中任一项所述的合成方法，其特征在于，所述步骤S6包括：采用醋酸水溶液对所述地加瑞克进行转盐，得到所述醋酸地加瑞克。The synthesis method according to any one of claims 1 to 4, wherein the step S6 comprises: using an aqueous acetic acid solution to salt the degarelix to obtain the degarelix acetate.
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