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WO2023030277A1 - Method for fully liquid-phase synthesis of grnh nonapeptide amide analog - Google Patents

Method for fully liquid-phase synthesis of grnh nonapeptide amide analog Download PDF

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Publication number
WO2023030277A1
WO2023030277A1 PCT/CN2022/115636 CN2022115636W WO2023030277A1 WO 2023030277 A1 WO2023030277 A1 WO 2023030277A1 CN 2022115636 W CN2022115636 W CN 2022115636W WO 2023030277 A1 WO2023030277 A1 WO 2023030277A1
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compound
tbu
reaction
leu
boc
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French (fr)
Chinese (zh)
Inventor
孙鹏程
潘静
吴军勇
唐勇擘
杜一雄
郭林
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Hunan Micro Peptide Biomedical Co Ltd
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Hunan Micro Peptide Biomedical Co Ltd
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Priority claimed from CN202111006173.0A external-priority patent/CN113603751A/en
Priority claimed from CN202111007826.7A external-priority patent/CN113527438A/en
Priority claimed from CN202111553517.XA external-priority patent/CN114149491A/en
Priority claimed from CN202111555663.6A external-priority patent/CN113968897A/en
Application filed by Hunan Micro Peptide Biomedical Co Ltd filed Critical Hunan Micro Peptide Biomedical Co Ltd
Publication of WO2023030277A1 publication Critical patent/WO2023030277A1/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
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • 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
    • C07K1/06—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
    • C07K1/08—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 using 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
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the technical field of pharmaceutical synthesis, in particular to a method for synthesizing GRnH nonapeptide amide analogues in full liquid phase.
  • Chinese patent CN201710851059.5 provides a solid-phase preparation method of alanorelin, which is costly, requires ammonolysis, difficult to control the reaction conditions, has many side reactions, and is not suitable for large-scale production.
  • Chinese patent CN202011087669.0 provides a method for synthesizing alanorelin by solid-liquid combination of polypeptides. This method is improved on the basis of the solid-phase method, but there is still the problem that impurities are difficult to purify.
  • Chinese patent CN201210334868.6 is a method for synthesizing histrelin.
  • the technical problem to be solved by the present invention is to overcome the disadvantages of the current mainstream solid-phase reaction, such as high cost, many solvents, precursor reagents, high pressure on environmental protection, and low purity of the crude product, thereby providing an all-liquid phase synthesis Methods for GRnH nonapeptide amide analogs.
  • This method synthesizes "Trp-Ser-Tyr” fragments, "R-Leu” fragments, “Pyr-His” fragments, and "Arg-Pro-NHEt” respectively, and obtains them through the "3+2+2+2" fragment condensation method GRnH nonapeptide amide analogs with high yield and high purity.
  • a method for the full liquid phase synthesis of GRnH nonapeptide amide analogues comprises the following steps:
  • R 1 is an amino protecting group, including any one of Fmoc, Z, Boc
  • R 2 is a carboxyl protecting group, including methyl ester Me, ethyl ester Et, benzyl ester Bzl, trityl ester Tr Any one
  • R3 includes any one of Boc or Trt
  • R4 is an amino protecting group, including any one of Fmoc, Z, Boc
  • R5 is D-Ala, D-Leu, D-Trp , any one of D-His.
  • the GRnH nonapeptide amide analogue when R 5 is a D-Ala group, the GRnH nonapeptide amide analogue is alanine; when R 5 is D-Leu, the GRnH nonapeptide amide analogue is leuprolide; when R 5 is During D-Trp, the GRnH nonapeptide amide analogue is deslorelin; when R 5 is D-His, the GRnH nonapeptide amide analogue is histrelin, especially, when R 5 is D-His, His also contains the protecting group Bzl.
  • step S1 specifically includes the following steps:
  • step S2 specifically includes the following steps:
  • Condensation reaction is carried out with R 1 -R 5 -OH, H-Leu-OR 2 as the reaction unit, the molar ratio of R 1 -R 5 -OH to H-Leu-OR 2 is 1:1.05-2, adding activator, Organic base, condensing agent, the ratio of H-Leu-OR 2 to activator, condensing agent, and organic base is 1:1:1:1, after the reaction is complete, filter, precipitate, wash, dry, and collect the solid to obtain compound 2;
  • the activator is a commonly used activator for polypeptide synthesis, including any one of HOSu, HOBt, HOAt, HOOBt;
  • the condensing agent is a commonly used condensing agent for polypeptide synthesis, including DCC, DIC, EDC, BOP, pyBOP, AOP , TBTU, HBTU, HATU;
  • the organic base includes any one of DIEA, TEA, NMM;
  • the solvent includes any one of THF, DCM, DMF, NMP, dioxane.
  • step S3 specifically includes the following steps:
  • step S2 Using compound 2 prepared in step S2 as a substrate, adding a deprotection reagent and a solvent, concentrating to a small amount, separating out, filtering, and drying in vacuo to obtain compound 3;
  • the deprotection reagent includes any one of trifluoroacetic acid, diethylamine, piperazine, and piperidine; the solvent is any one of DMF, methanol, ethanol, DCM, and THF.
  • step S4 specifically includes the following steps:
  • Condensation reaction is carried out with compound 1 synthesized in step S1 and compound 3 synthesized in step S3 as reaction units, wherein the molar ratio of compound 1 and compound 3 is 1: 1.05-2, adding organic base and condensing agent, wherein compound 3 and organic base 1.
  • the molar ratio of the condensing agent is 1:1:1.
  • the condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU;
  • the organic base includes any one of DIEA, TEA, and NMM species;
  • the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.
  • step S5 specifically includes the following steps:
  • the deprotection reagent includes any one of diethylamine, piperazine, and piperidine solutions .
  • step S6 specifically includes the following steps:
  • Condensation reaction is carried out with Fmoc-His(R 3 )-OH and compound 5 synthesized in step S5 as the reaction unit, wherein the molar ratio of compound 5 to Fmoc-His(R 3 )-OH is 1:1.05-2, adding activating agent, organic base, condensing agent, wherein the ratio of Fmoc-His(R 3 )-OH to activator, condensing agent, and organic base is 1:1:1:1, the reaction is complete in the solvent, concentrated, filtered, washed, Dry to obtain compound 6;
  • the activator is a commonly used activator for polypeptide synthesis, including any one of HOSu, HOBt, HOAt, HOOBt;
  • the condensing agent is a commonly used condensing agent for polypeptide synthesis, including DCC, DIC, EDC, BOP, pyBOP, AOP , TBTU, HBTU, HATU;
  • the organic base includes any one of DIEA, TEA, NMM;
  • the solvent includes any one of THF, DCM, DMF, NMP, dioxane.
  • step S7 specifically includes the following steps:
  • step S6 Perform condensation reaction with R 4 -Pyr-OH and compound 6 synthesized in step S6, wherein the molar ratio of compound 6 to R 4 -Pyr-OH is 1:1.05-2; add organic base and condensing agent, wherein R 4 -Pyr The molar ratio of -OH to condensing agent and organic base is 1:1:1. After the reaction is complete, filter, wash, and dry to obtain compound 7;
  • the condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU;
  • the organic base includes any one of DIEA, TEA, and NMM species;
  • the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.
  • step S8 specifically includes the following steps:
  • the molar ratio of NaOH and compound 7 is 1.5:1-20:1;
  • Step S9 specifically includes the following steps:
  • the condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU;
  • the organic base includes any one of DIEA, TEA, and NMM species;
  • the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.
  • step S10 specifically includes the following steps:
  • the condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU;
  • the organic base includes any one of DIEA, TEA, and NMM A kind;
  • Described solvent comprises any one in THF, DCM, DMF, NMP, dioxane;
  • Step S11 specifically includes the following steps:
  • reagents used in the above-mentioned technical schemes are common commercially available medicaments; in the above-mentioned technical schemes, ether reagents are usually used for precipitation or solid precipitation operations, including any one of petroleum ether, isopropyl ether, diethyl ether or Any combination, preferably, petroleum ether.
  • the invention creatively invents a green and mild production process through the full liquid phase synthesis method, without using any highly toxic and precursor reagents, greatly reducing the cost, and is very suitable for large-scale production.
  • the yield of the crude histrelin produced by the method provided by the invention is more than 85%, and the purity of the crude product can reach more than 80%; the purity of the crude product of alanorelin can reach more than 95%; It can reach more than 90%.
  • Fig. 1 is the HPLC spectrogram of the amolide crude product prepared in Example 1 of the present invention.
  • Fig. 2 is the HPLC spectrogram of the leuprolide crude product prepared in Example 3 of the present invention.
  • Fig. 3 is the HPLC spectrogram of the crude deslorelin prepared in Example 4 of the present invention.
  • Fig. 4 is the HPLC spectrogram of the crude histrelin prepared in Example 5 of the present invention.
  • HBTU O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • HATU O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • DIEA N,N-Diisopropylethylamine NMM N-methylmorphine
  • a method for the synthesis of Amolide by full liquid phase method comprising the following steps:
  • the reaction solution was poured into the Erlenmeyer flask twice, and then 0.5M hydrochloric acid was added to rapidly stir and precipitate, and the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solids into containers and weigh.
  • the HPLC detection condition of compound 1 is:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • reaction solution was filtered and precipitated with 0.5M hydrochloric acid aqueous solution.
  • the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solid and put it in the Erlenmeyer flask and weigh it; the HPLC analysis conditions of compound 2 are:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection condition of compound 6 is:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Boc-Pyr-OH (74.5mmol) and BOP (74.5mmol) in a reaction flask, dissolve them completely in DMF, and then add DIEA (74.5mmol) in a cold bath for 10 minutes, then add H-His(Trt)-Trp( Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe (67.7mmol) was dissolved in DMF and added to the reaction to start the reaction.
  • HPLC detection conditions are as follows:
  • Mobile phase A 0.1% TFA/water
  • Mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection conditions are as follows:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection conditions are as follows:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection condition is:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • a method for the synthesis of Amolide by full liquid phase method comprising the following steps:
  • HPLC detection conditions were the same as in Example 1.
  • a method for synthesizing deslorelin by a full liquid phase method comprising the following steps:
  • the reaction solution was poured into the Erlenmeyer flask twice, and then 0.5M hydrochloric acid was added to rapidly stir and precipitate, and the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solids into containers and weigh.
  • the HPLC detection condition of compound 1 is:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • reaction solution was filtered and precipitated with 0.5M hydrochloric acid aqueous solution.
  • the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solid and put it in the Erlenmeyer flask and weigh it; the HPLC analysis conditions of compound 2 are:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection condition of compound 6 is:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Boc-Pyr-OH (81.2mmol) and BOP (81.2mmol) in a reaction flask, dissolve them completely in DMF, then add DIEA (81.2mmol) in a cold bath for 10min, then add H-His(Trt)-Trp( Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe (73.8mmol) was dissolved in DMF and added to the reaction to start the reaction.
  • HPLC detection conditions are as follows:
  • Mobile phase A 0.1% TFA/water
  • Mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection conditions are as follows:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection conditions are as follows:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection conditions are:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • a method for synthesizing leuprolide by a full liquid phase method comprising the following steps:
  • the reaction solution was poured into the Erlenmeyer flask twice, and then 0.5M hydrochloric acid was added to rapidly stir and precipitate, and the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solids into containers and weigh.
  • the HPLC detection condition of compound 1 is:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • reaction solution was filtered and precipitated with 0.5M hydrochloric acid aqueous solution.
  • the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solid and put it in the Erlenmeyer flask and weigh it; the HPLC analysis conditions of compound 2 are:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection condition of compound 6 is:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Boc-Pyr-OH (76.3mmol) and BOP (76.3mmol) in the reaction flask dissolve them completely in DMF, then add DIEA (76.3mmol) in the cold bath for 10min, then add H-His(Trt)-Trp( Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe (69.4mmol) was dissolved in DMF and added to the reaction to start the reaction.
  • HPLC detection conditions are as follows:
  • Mobile phase A 0.1% TFA/water
  • Mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection conditions are as follows:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection conditions are as follows:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection conditions are:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • a method for synthesizing histrelin by a full liquid phase method comprising the following steps:
  • the reaction solution was poured into the Erlenmeyer flask twice, and then 0.5M hydrochloric acid was added to rapidly stir and precipitate, and the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solids into containers and weigh.
  • the HPLC detection condition of compound 1 is:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • reaction solution was filtered and precipitated with 0.5M hydrochloric acid aqueous solution.
  • the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solid and put it in the Erlenmeyer flask and weigh it; the HPLC analysis conditions of compound 2 are:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection condition of compound 6 is:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • Boc-Pyr-OH (76.3mmol) and BOP (76.3mmol) in the reaction flask dissolve them completely in DMF, then add DIEA (76.3mmol) in the cold bath for 10min, then add H-His(Trt)-Trp( Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe(69.4mmol) was dissolved in DMF and added to the reaction to start the reaction.
  • HPLC detection conditions are as follows:
  • Mobile phase A 0.1% TFA/water
  • Mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection conditions are as follows:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection conditions are as follows:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
  • HPLC detection conditions are:
  • Mobile phase A 0.1% TFA/water
  • mobile phase B 0.1% TFA/acetonitrile
  • Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,

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Abstract

Provided is a method for fully liquid-phase synthesis of a GRnH nonapeptide amide analog, which belongs to the technical field of medicine synthesis. The method comprises respectively synthesizing a "Trp-Ser-Tyr" fragment, an "R-Leu" fragment, a "Pyr-His" fragment and "Arg-Pro-Hunan T", wherein, R = D-Ala (alarelin), D-Leu (leuprorelin), D-Trp (deslorelin) and D-His (histrelin), and obtaining the GRnH nonapeptide amide analog with high yield and high purity by means of a "3 +2 +2 +2" fragment condensation method. The synthesis method is environmentally friendly, mild, and free of any highly toxic and poisonable reagents. The cost is greatly reduced and the synthesis method is very suitable for large-scale production.

Description

一种全液相合成GRnH九肽酰胺类似物的方法A kind of method of whole liquid phase synthesis GRnH nonapeptide amide analogue 技术领域technical field

本发明涉及医药合成技术领域，具体涉及一种全液相合成GRnH九肽酰胺类似物的方法。The invention relates to the technical field of pharmaceutical synthesis, in particular to a method for synthesizing GRnH nonapeptide amide analogues in full liquid phase.

背景技术Background technique

人工合成的促性腺激素释放激素(GnRH)的九肽酰胺类似物有丙氨瑞林、地洛瑞林、亮丙瑞林、组氨瑞林等，可用于治疗中枢性性早熟症。其氨基酸序列为：Pyr-His-Trp-Ser-Tyr-R-Leu-Arg-Pro-NHEt，R＝D-Ala(丙氨瑞林)、D-Leu(亮丙瑞林)、D-Trp(地洛瑞林)、D-His(组氨瑞林)。Synthetic nonapeptide amide analogues of gonadotropin-releasing hormone (GnRH) include alanorelin, deslorelin, leuprolide, histrelin, etc., which can be used to treat central precocious puberty. Its amino acid sequence is: Pyr-His-Trp-Ser-Tyr-R-Leu-Arg-Pro-NHEt, R=D-Ala (alanamine), D-Leu (leuprolide), D-Trp (Deslorelin), D-His (Histrelin).

现有技术中，GRnH九肽酰胺类似物主要合成方法为固相法。如中国专利CN201710851059.5提供了一种丙氨瑞林的固相制备方法，该法成本高，需要氨解，反应条件难以控制，副反应较多，不适合规模化生产。中国专利CN202011087669.0提供了一种多肽固液组合合成丙氨瑞林的方法，该法在固相法的基础上进行了改进，但还是存在杂质难以纯化的问题。中国专利CN201210334868.6一种组氨瑞林的合成方法，该法需要在树脂上进行接肽操作，产品收率低，纯度低，不适合大规模生产。亮丙瑞林的合成工艺如EP1088555、EP1777232、US5480868、CN1865280等。固相合成需要使用昂贵的树脂，这不仅给企业的大规模生产带来成本的压力，而且使用溶剂多，使用易制毒试剂，环保压力大，而且在最后切肽的工序中，叔丁基很容易在酸性条件下被脱除，生成其他杂质产物，导致产品粗品纯度低。目前，还未有全液相合成GRnH九肽酰胺类似物的报道。In the prior art, the main synthesis method of GRnH nonapeptide amide analogs is the solid-phase method. For example, Chinese patent CN201710851059.5 provides a solid-phase preparation method of alanorelin, which is costly, requires ammonolysis, difficult to control the reaction conditions, has many side reactions, and is not suitable for large-scale production. Chinese patent CN202011087669.0 provides a method for synthesizing alanorelin by solid-liquid combination of polypeptides. This method is improved on the basis of the solid-phase method, but there is still the problem that impurities are difficult to purify. Chinese patent CN201210334868.6 is a method for synthesizing histrelin. This method needs to carry out peptide grafting operation on the resin, the product yield is low, the purity is low, and it is not suitable for large-scale production. The synthetic technique of leuprolide is as EP1088555, EP1777232, US5480868, CN1865280 etc. Solid-phase synthesis requires the use of expensive resins, which not only brings cost pressures to the large-scale production of enterprises, but also uses many solvents and precursor reagents, which puts a lot of pressure on environmental protection, and in the final peptide cutting process, tert-butyl It is easy to be removed under acidic conditions to generate other impurity products, resulting in low purity of the crude product. At present, there is no report on the synthesis of GRnH nonapeptide amide analogues in full solution phase.

发明内容Contents of the invention

因此，本发明要解决的技术问题在于克服了目前主流的固相反应成本高，使用溶剂多，使用易制毒试剂、环保压力大，产品粗品纯度低的缺点，从而提供一种全液相合成GRnH九肽酰胺类似物的方法。该方法分别合成“Trp-Ser-Tyr”片段、“R-Leu”片段、“Pyr-His”片段、“Arg-Pro-NHEt”，通过“3+2+2+2”的片段缩合方式得到收率高、纯度高的GRnH九肽酰胺类似物。Therefore, the technical problem to be solved by the present invention is to overcome the disadvantages of the current mainstream solid-phase reaction, such as high cost, many solvents, precursor reagents, high pressure on environmental protection, and low purity of the crude product, thereby providing an all-liquid phase synthesis Methods for GRnH nonapeptide amide analogs. This method synthesizes "Trp-Ser-Tyr" fragments, "R-Leu" fragments, "Pyr-His" fragments, and "Arg-Pro-NHEt" respectively, and obtains them through the "3+2+2+2" fragment condensation method GRnH nonapeptide amide analogs with high yield and high purity.

本发明提供的一种全液相合成GRnH九肽酰胺类似物的方法，包括如下步骤：A method for the full liquid phase synthesis of GRnH nonapeptide amide analogues provided by the invention comprises the following steps:

S1、液相合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH；S1. Compound 1 synthesized in liquid phase: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH;

S2、液相合成化合物2：R 1-R 5-Leu-OR 2； S2, liquid phase synthesis of compound 2: R 1 -R 5 -Leu-OR 2 ;

S3、液相合成化合物3：H-R 5-Leu-OR 2； S3, liquid phase synthesis of compound 3: HR 5 -Leu-OR 2 ;

S4、液相合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OR 2； S4, liquid phase synthesis of compound 4: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ;

S5、液相合成化合物5：H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OR 2； S5, liquid phase synthesis of compound 5: H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ;

S6、液相合成化合物6：H-His(R 3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OR 2； S6, liquid phase synthesis of compound 6: H-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ;

S7、液相合成化合物7：S7, liquid phase synthesis of compound 7:

R 4-Pyr-His(R 3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OR 2； R 4 -Pyr-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ;

S8、液相合成化合物8：S8, liquid phase synthesis of compound 8:

R 4-Pyr-His(R 3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OH； R 4 -Pyr-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OH;

S9、液相合成化合物9：H-Arg(pbf)-Pro-NHEt；S9, liquid phase synthesis of compound 9: H-Arg(pbf)-Pro-NHEt;

S10、液相合成化合物10：S10, liquid phase synthesis of compound 10:

R 4-Pyr-His(R 3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-Arg(pbf)-Pro-NHEt； R 4 -Pyr-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-Arg(pbf)-Pro-NHEt;

S11、GRnH九肽酰胺类似物粗品的制备；S11, preparation of crude product of GRnH nonapeptide amide analog;

其中，R 1为氨基保护基团，包括Fmoc、Z、Boc中的任意一种；R 2为羧基保护基团，包括甲酯Me，乙酯Et，苄酯Bzl，三苯甲酯Tr中的任意一种；R 3包括Boc或Trt中的任意一种；R 4为氨基保护基团，包括Fmoc、Z、Boc中的任意一种；R 5为D-Ala、D-Leu、D-Trp、D-His中的任意一种。例如，当R 5为D-Ala基团时，GRnH九肽酰胺类似物为丙氨瑞林；当R 5为D-Leu时，GRnH九肽酰胺类似物为亮丙瑞林；当R 5为D-Trp时、GRnH九肽酰胺类似物为地洛瑞林；当R 5为D-His时，GRnH九肽酰胺类似物为组氨瑞林，特别地，当R 5为D-His时，His还包含保护基团Bzl。 Wherein, R 1 is an amino protecting group, including any one of Fmoc, Z, Boc; R 2 is a carboxyl protecting group, including methyl ester Me, ethyl ester Et, benzyl ester Bzl, trityl ester Tr Any one; R3 includes any one of Boc or Trt; R4 is an amino protecting group, including any one of Fmoc, Z, Boc; R5 is D-Ala, D-Leu, D-Trp , any one of D-His. For example, when R 5 is a D-Ala group, the GRnH nonapeptide amide analogue is alanine; when R 5 is D-Leu, the GRnH nonapeptide amide analogue is leuprolide; when R 5 is During D-Trp, the GRnH nonapeptide amide analogue is deslorelin; when R 5 is D-His, the GRnH nonapeptide amide analogue is histrelin, especially, when R 5 is D-His, His also contains the protecting group Bzl.

在较优的技术方案中，步骤S1具体包括以下步骤：In a preferred technical solution, step S1 specifically includes the following steps:

以Fmoc-Trp(Boc)-Ser(tBu)-OSu和H-Tyr(tBu)-OH为反应单元进行缩合反应，在溶剂中反应得到化合物1；所述Fmoc-Trp(Boc)-Ser(tBu)-OSu和所述H-Tyr(tBu)-OH的摩尔比为1∶1.05-2，所述H-Tyr(tBu)-OH与所述有机碱的摩尔比为1∶1，所述溶剂包括DMF、THF、甲醇、乙醇、NMP中的任意一种。Take Fmoc-Trp(Boc)-Ser(tBu)-OSu and H-Tyr(tBu)-OH as reaction units to carry out condensation reaction, react in a solvent to obtain compound 1; said Fmoc-Trp(Boc)-Ser(tBu )-OSu and the H-Tyr(tBu)-OH molar ratio is 1:1.05-2, the H-Tyr(tBu)-OH and the organic base molar ratio is 1:1, the solvent Including any one of DMF, THF, methanol, ethanol, and NMP.

在较优的技术方案中，步骤S2具体包括以下步骤：In a preferred technical solution, step S2 specifically includes the following steps:

以R 1-R 5-OH、H-Leu-OR 2为反应单元进行缩合反应，R 1-R 5-OH与H-Leu-OR 2的摩尔比为1∶1.05-2，加入活化剂、有机碱、缩合剂，H-Leu-OR 2与活化剂、缩 合剂、有机碱的比为1∶1∶1∶1，反应完全后，过滤、析出、洗涤、干燥，收集固体得化合物2； Condensation reaction is carried out with R 1 -R 5 -OH, H-Leu-OR 2 as the reaction unit, the molar ratio of R 1 -R 5 -OH to H-Leu-OR 2 is 1:1.05-2, adding activator, Organic base, condensing agent, the ratio of H-Leu-OR 2 to activator, condensing agent, and organic base is 1:1:1:1, after the reaction is complete, filter, precipitate, wash, dry, and collect the solid to obtain compound 2;

所述活化剂为多肽合成常用的活化剂，包括HOSu、HOBt、HOAt、HOOBt中的任意一种；所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The activator is a commonly used activator for polypeptide synthesis, including any one of HOSu, HOBt, HOAt, HOOBt; the condensing agent is a commonly used condensing agent for polypeptide synthesis, including DCC, DIC, EDC, BOP, pyBOP, AOP , TBTU, HBTU, HATU; the organic base includes any one of DIEA, TEA, NMM; the solvent includes any one of THF, DCM, DMF, NMP, dioxane.

在较优的技术方案中，步骤S3具体包括以下步骤：In a preferred technical solution, step S3 specifically includes the following steps:

以步骤S2制得的化合物2为底物，加入脱保护试剂和溶剂，浓缩至少量，析出，过滤，真空干燥得化合物3；Using compound 2 prepared in step S2 as a substrate, adding a deprotection reagent and a solvent, concentrating to a small amount, separating out, filtering, and drying in vacuo to obtain compound 3;

所述脱保护试剂包括三氟乙酸、二乙胺、哌嗪、哌啶中的任意一种；所述溶剂为DMF、甲醇、乙醇、DCM、THF中的任意一种。The deprotection reagent includes any one of trifluoroacetic acid, diethylamine, piperazine, and piperidine; the solvent is any one of DMF, methanol, ethanol, DCM, and THF.

在较优的技术方案中，步骤S4具体包括以下步骤：In a preferred technical solution, step S4 specifically includes the following steps:

以步骤S1合成的化合物1和步骤S3合成的化合物3为反应单元进行缩合反应，其中化合物1和化合物3的摩尔比为1∶1.05-2，加入有机碱、缩合剂，其中化合物3与有机碱、缩合剂的摩尔比为1∶1∶1，在溶剂中反应完全后，浓缩、过滤、洗涤、干燥，得化合物4；Condensation reaction is carried out with compound 1 synthesized in step S1 and compound 3 synthesized in step S3 as reaction units, wherein the molar ratio of compound 1 and compound 3 is 1: 1.05-2, adding organic base and condensing agent, wherein compound 3 and organic base 1. The molar ratio of the condensing agent is 1:1:1. After the reaction in the solvent is complete, concentrate, filter, wash, and dry to obtain compound 4;

所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM species; the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.

在较优的技术方案中，步骤S5中具体包括以下步骤：In a preferred technical solution, step S5 specifically includes the following steps:

取化合物4，加入脱保护试剂反应脱去Fmoc基团，浓缩至少量，析出固体，过滤，真空干燥得化合物5；所述脱保护试剂包括二乙胺、哌嗪、哌啶溶液的任意一种。Take compound 4, add a deprotection reagent to react to remove the Fmoc group, concentrate to a certain amount, precipitate a solid, filter, and vacuum-dry to obtain compound 5; the deprotection reagent includes any one of diethylamine, piperazine, and piperidine solutions .

在较优的技术方案中，步骤S6具体包括以下步骤：In a preferred technical solution, step S6 specifically includes the following steps:

以Fmoc-His(R 3)-OH、步骤S5中合成的化合物5为反应单元进行缩合反应，其中化合物5与Fmoc-His(R 3)-OH的摩尔比为1∶1.05-2，加入活化剂、有机碱、缩合剂，其中Fmoc-His(R 3)-OH与活化剂、缩合剂、有机碱的比为1∶1∶1∶1，在溶剂中反应完全，浓缩、过滤、洗涤、干燥，得化合物6； Condensation reaction is carried out with Fmoc-His(R 3 )-OH and compound 5 synthesized in step S5 as the reaction unit, wherein the molar ratio of compound 5 to Fmoc-His(R 3 )-OH is 1:1.05-2, adding activating agent, organic base, condensing agent, wherein the ratio of Fmoc-His(R 3 )-OH to activator, condensing agent, and organic base is 1:1:1:1, the reaction is complete in the solvent, concentrated, filtered, washed, Dry to obtain compound 6;

所述活化剂为多肽合成常用的活化剂，包括HOSu、HOBt、HOAt、HOOBt中的 任意一种；所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The activator is a commonly used activator for polypeptide synthesis, including any one of HOSu, HOBt, HOAt, HOOBt; the condensing agent is a commonly used condensing agent for polypeptide synthesis, including DCC, DIC, EDC, BOP, pyBOP, AOP , TBTU, HBTU, HATU; the organic base includes any one of DIEA, TEA, NMM; the solvent includes any one of THF, DCM, DMF, NMP, dioxane.

在较优的技术方案中，步骤S7具体包括以下步骤：In a preferred technical solution, step S7 specifically includes the following steps:

以R 4-Pyr-OH和步骤S6合成的化合物6进行缩合反应，其中化合物6与R 4-Pyr-OH的摩尔比为1∶1.05-2；加入有机碱、缩合剂，其中R 4-Pyr-OH与缩合剂、有机碱的摩尔比为1∶1∶1，反应完全后，过滤、洗涤、干燥，得化合物7； Perform condensation reaction with R 4 -Pyr-OH and compound 6 synthesized in step S6, wherein the molar ratio of compound 6 to R 4 -Pyr-OH is 1:1.05-2; add organic base and condensing agent, wherein R 4 -Pyr The molar ratio of -OH to condensing agent and organic base is 1:1:1. After the reaction is complete, filter, wash, and dry to obtain compound 7;

所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM species; the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.

在较优的技术方案中，步骤S8具体包括以下步骤：In a preferred technical solution, step S8 specifically includes the following steps:

取甲醇和化合物7反应，缓慢加入2M NaOH，反应2-4h，过滤、洗涤、干燥得化合物8；Take methanol and compound 7 to react, slowly add 2M NaOH, react for 2-4h, filter, wash and dry to obtain compound 8;

其中，NaOH和化合物7的摩尔比为1.5∶1-20∶1；Wherein, the molar ratio of NaOH and compound 7 is 1.5:1-20:1;

步骤S9具体包括以下步骤：Step S9 specifically includes the following steps:

以R 1-Arg(pbf)-OH、H-Pro-NHEt.HCl为反应单元进行缩合反应，其中R 1-Arg(pbf)-OH和H-Pro-NHEt.HCl的摩尔比为1∶1.05-2，加入有机碱、缩合剂，其中H-Pro-NHEt.HCl与有机碱、缩合剂的摩尔比为1∶1∶1，在溶剂中反应完全后，析出固体，过滤，干燥，脱保护，浓缩，析出固体，过滤，真空干燥得化合物9； Condensation reaction with R 1 -Arg(pbf)-OH, H-Pro-NHEt.HCl as reaction units, wherein the molar ratio of R 1 -Arg(pbf)-OH and H-Pro-NHEt.HCl is 1:1.05 -2. Add organic base and condensing agent, wherein the molar ratio of H-Pro-NHEt.HCl to organic base and condensing agent is 1:1:1. After complete reaction in the solvent, a solid is precipitated, filtered, dried, and deprotected , concentrated, precipitated solid, filtered, and dried in vacuo to obtain compound 9;

所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM species; the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.

在较优的技术方案中，步骤S10具体包括以下步骤：In a preferred technical solution, step S10 specifically includes the following steps:

以化合物8和化合物9为反应单元进行缩合反应，其中化合物8和化合物9的摩尔比为1∶1.05-2，加入缩合剂、有机碱，其中化合物9和缩合剂、有机碱的摩尔比为1∶1∶1，反应完全后，过滤、洗涤、干燥得化合物10；Carry out condensation reaction with compound 8 and compound 9 as the reaction unit, wherein the molar ratio of compound 8 and compound 9 is 1: 1.05-2, add condensing agent, organic base, wherein the molar ratio of compound 9 and condensing agent, organic base is 1 : 1:1, after the reaction is complete, filter, wash, and dry to obtain compound 10;

所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的 任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种；The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM A kind; Described solvent comprises any one in THF, DCM, DMF, NMP, dioxane;

步骤S11具体包括以下步骤：Step S11 specifically includes the following steps:

取化合物10于反应器中，加入裂解液反应完成后，用冷冻乙醚沉淀，过滤，收集固体，得到GRnH九肽酰胺类似物粗品；Take compound 10 in the reactor, add the lysate and after the reaction is completed, precipitate with frozen ether, filter, collect the solid, and obtain the crude product of GRnH nonapeptide amide analogue;

所述裂解液的组分，按体积比计，包括：TFA∶TIS∶H 2O＝95∶2.5∶2.5。 The components of the lysate, by volume ratio, include: TFA:TIS:H 2 O=95:2.5:2.5.

需要说明的是，上述技术方案中所用试剂均为普通市售药剂；在上述技术方案中，析出或者析出固体操作通常采用醚类试剂，包括石油醚、异丙醚、乙醚中的任意一种或任意组合，优选地，为石油醚。It should be noted that the reagents used in the above-mentioned technical schemes are common commercially available medicaments; in the above-mentioned technical schemes, ether reagents are usually used for precipitation or solid precipitation operations, including any one of petroleum ether, isopropyl ether, diethyl ether or Any combination, preferably, petroleum ether.

本发明技术方案，具有如下优点：The technical solution of the present invention has the following advantages:

本发明通过全液相合成法，创造性地发明绿色、温和的生产工艺，没有使用任何剧毒、易制毒试剂，成本极大降低，非常适合大规模生产。The invention creatively invents a green and mild production process through the full liquid phase synthesis method, without using any highly toxic and precursor reagents, greatly reducing the cost, and is very suitable for large-scale production.

本发明提供的方法生产的组氨瑞林粗品收率在85％以上，粗品纯度可达80％以上；丙氨瑞林粗品纯度可达95％以上；亮丙瑞林、地洛瑞林粗品纯度可达90％以上。The yield of the crude histrelin produced by the method provided by the invention is more than 85%, and the purity of the crude product can reach more than 80%; the purity of the crude product of alanorelin can reach more than 95%; It can reach more than 90%.

附图说明Description of drawings

为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work.

图1本发明实施例1制备的丙氨瑞林粗品的HPLC谱图。Fig. 1 is the HPLC spectrogram of the amolide crude product prepared in Example 1 of the present invention.

图2本发明实施例3制备的亮丙瑞林粗品的HPLC谱图。Fig. 2 is the HPLC spectrogram of the leuprolide crude product prepared in Example 3 of the present invention.

图3本发明实施例4制备的地洛瑞林粗品的HPLC谱图。Fig. 3 is the HPLC spectrogram of the crude deslorelin prepared in Example 4 of the present invention.

图4本发明实施例5制备的组氨瑞林粗品的HPLC谱图。Fig. 4 is the HPLC spectrogram of the crude histrelin prepared in Example 5 of the present invention.

具体实施方式Detailed ways

本发明权利要求书和说明书中出现物质的英文缩写对应的中文名称见表1。See Table 1 for the Chinese names corresponding to the English abbreviations of the substances appearing in the claims of the present invention and the description.

表1Table 1

英文缩写English abbreviations 中文名称Chinese name FmocFmoc 9-芴甲氧羰基9-fluorenylmethoxycarbonyl TrpTrp 色氨酸Tryptophan

BocBoc 叔丁氧羰基tert-butoxycarbonyl SerSer 丝氨酸serine tButB 叔丁基tert-butyl TyrTyr 酪氨酸Tyrosine AlaAla 丙氨酸Alanine LeuLeu 亮氨酸Leucine ArgArg 精氨酸arginine pbfpbf 2，2，4，6，7-五甲基二氢苯并呋喃基2,2,4,6,7-Pentamethyldihydrobenzofuryl ProPro 脯氨酸proline EtEt 乙基Ethyl PyrPyr 焦谷氨酸pyroglutamic acid HisHis 组氨酸Histidine DMFDMF N，N-二甲基甲酰胺N,N-Dimethylformamide THFTHF 四氢呋喃Tetrahydrofuran HOSUHOSU 琥珀酰亚胺Succinimide DCCDCC 二环己基碳二亚胺Dicyclohexylcarbodiimide TEATEA 三乙胺Triethylamine DEADEA 二乙胺Diethylamine TFATFA 三氟乙酸Trifluoroacetate HOBtHOB 1-羟基苯并三唑1-Hydroxybenzotriazole HOAtHOAt N-羟基-7-氮杂苯并三氮唑N-Hydroxy-7-azabenzotriazole HOOBtHOOB 3，4-二氢-3-羟基-4-氧-1，2，3-苯并三嗪3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine DICDIC N，N′-二异丙基碳二亚胺N,N'-Diisopropylcarbodiimide EDCEDC 1，2-二氯乙烷1,2-Dichloroethane BOPBOP 卡特缩合剂Carter condensation agent pyBOPpyBOP 1H-苯并三唑-1-基氧三吡咯烷基六氟磷酸盐1H-Benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate AOPAOP 7-氮杂苯并三唑-1-基氧基三(二甲胺基)膦六氟磷酸盐7-Azabenzotriazol-1-yloxytris(dimethylamino)phosphine hexafluorophosphate TBTUTBTU 2-(1H-苯并三偶氮L-1-基)-1，1，3，3-四甲基脲四氟硼酸酯2-(1H-Benzotrisazo L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

HBTUHBTU O-(苯并三唑-1-基)-N，N，N′，N′-四甲基脲六氟磷酸酯O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATUHATU O-(7-氮杂苯并三唑-1-基)-N，N，N′，N′-四甲基脲六氟磷酸盐O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate DIEADIEA N，N-二异丙基乙胺N,N-Diisopropylethylamine NMMNMM N-甲基吗啡林N-methylmorphine DCMDCM 二氯甲烷Dichloromethane NMPNMP N-甲基-2-吡咯烷酮N-methyl-2-pyrrolidone ZZ 苄氧酰基Benzyloxyacyl

实施例1Example 1

一种全液相法合成丙氨瑞林的方法，包括以下步骤：A method for the synthesis of Amolide by full liquid phase method, comprising the following steps:

1、合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH1. Synthesis of compound 1: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH

将Fmoc-Trp(Boc)-Ser(tBu)-OSu(100mmol)用DMF完全溶解后，再准确称取H-Tyr(tBu)-OH(110mmol)加入上述反应瓶，加TEA(110mmol)开始反应。Dissolve Fmoc-Trp(Boc)-Ser(tBu)-OSu(100mmol) completely in DMF, then accurately weigh H-Tyr(tBu)-OH(110mmol) into the above reaction flask, add TEA(110mmol) to start the reaction .

搅拌反应60min后，HPLC检测反应完全。After stirring and reacting for 60 min, HPLC detected that the reaction was complete.

将反应液分两次倒入三角瓶中，再加入0.5M盐酸快速搅拌析出，过滤的固体，然后用纯化水洗至中性，30℃干燥。收集固体装至容器中，称重。化合物1的HPLC检测条件为：The reaction solution was poured into the Erlenmeyer flask twice, and then 0.5M hydrochloric acid was added to rapidly stir and precipitate, and the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solids into containers and weigh. The HPLC detection condition of compound 1 is:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000001
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000001

梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.

收率：98％；纯度：87.6％。Yield: 98%; Purity: 87.6%.

2、合成化合物2：Fmoc-D-Ala-Leu-OMe.HCl2. Synthesis of compound 2: Fmoc-D-Ala-Leu-OMe.HCl

准确称取Fmoc-D-Ala-OH(150mmol)、HOSU(165mmol)于反应瓶中，用DMF完全溶解后，再称取H-Leu-OMe.HCl(165mmol)于三角瓶中，用DMF完全溶解后冷浴10min，加入TEA(165mmol)，快速摇匀，加入上述反应瓶中，继续冷浴5min后加入DCC(165mmol)开始反应。反应1.5h后HPLC检测反应完全。Accurately weigh Fmoc-D-Ala-OH (150mmol) and HOSU (165mmol) in a reaction flask, dissolve them completely with DMF, then weigh H-Leu-OMe.HCl (165mmol) in a conical flask, Cool bath for 10 min after dissolving, add TEA (165 mmol), shake quickly, add to the above reaction flask, continue cooling bath for 5 min, then add DCC (165 mmol) to start the reaction. After 1.5 h of reaction, HPLC detected that the reaction was complete.

待完全反应后过滤反应液，用0.5M盐酸水溶液析出，过滤的固体，然后用纯化水洗至中性，30℃干燥。收集固体装至三角瓶中，称重；化合物2的HPLC 分析条件为：After the reaction was complete, the reaction solution was filtered and precipitated with 0.5M hydrochloric acid aqueous solution. The filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solid and put it in the Erlenmeyer flask and weigh it; the HPLC analysis conditions of compound 2 are:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000002
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000002

梯度：0-30min，50％B-90％B。Gradient: 0-30min, 50%B-90%B.

收率：103.6％，纯度：93.4％。Yield: 103.6%, purity: 93.4%.

3、合成化合物3：H-D-Ala-Leu-OMe3. Synthesis of compound 3: H-D-Ala-Leu-OMe

准确称取Fmoc-D-Ala-Leu-OMe.HCl(150mmol)于反应瓶中，加入二乙胺400ml反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。化合物3的HPLC检测条件为：Accurately weigh Fmoc-D-Ala-Leu-OMe.HCl (150mmol) into a reaction flask, add 400ml of diethylamine to react for 20min, concentrate to a small amount, add petroleum ether to precipitate a solid, filter, and vacuum dry. The HPLC detection condition of compound 3 is:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000003
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000003

梯度：0-30min，10％B-50％B。Gradient: 0-30min, 10%B-50%B.

收率：85.5％，纯度：91％。Yield: 85.5%, purity: 91%.

4、合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe4. Synthesis of compound 4: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe

准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH(98mmol)、BOP(107.8mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(107.8mmol)，再将H-D-Ala-Leu-OMe(107.8mmol)用DCM溶解后加入反应中开始反应。Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH(98mmol) and BOP(107.8mmol) in the reaction flask, dissolve them completely in DMF, and add DIEA(107.8mmol) in the cooling bath for 10min , and then H-D-Ala-Leu-OMe (107.8mmol) was dissolved in DCM and added to the reaction to start the reaction.

反应1.0h后HPLC检测反应完全，浓缩，用0.5M盐酸沉淀，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，称重；化合物4的HPLC检测条件为：After reacting for 1.0 h, HPLC detected that the reaction was complete, concentrated, precipitated with 0.5M hydrochloric acid, collected the solid by filtration, washed with purified water to neutral (pH test paper detection), and weighed; the HPLC detection conditions of compound 4 were:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000004
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000004

梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.

收率：95％，纯度：80％。Yield: 95%, purity: 80%.

5、合成化合物5：H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe5. Synthesis of compound 5: H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe

准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe(93.1mmol)于反应瓶中，加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。化合物5的HPLC分析条件为：Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe (93.1mmol) in a reaction bottle, add 500mL of diethylamine to react for 20min, concentrate to a certain amount, add petroleum Ether precipitated as a solid, which was filtered and dried in vacuo. The HPLC analysis conditions of compound 5 are:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000005
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000005

梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.

收率：91％，纯度：85.4％。Yield: 91%, purity: 85.4%.

6、合成化合物6：H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe6. Synthesis of compound 6: H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe

准确称取H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe(84.7mmol)、Fmoc-His(Trt)-OH(93.2mmol)、HOBt(93.2mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DCC(93.2mmol)开始反应。Accurately weigh H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe(84.7mmol), Fmoc-His(Trt)-OH(93.2mmol), HOBt(93.2mmol) In the reaction bottle, after completely dissolving with DMF, add DCC (93.2 mmol) to start the reaction after cooling for 10 min.

反应1.5h后HPLC检测反应完全，过滤反应液，倒入反应瓶中，再加入二乙胺反应20min，浓缩至少量，加入0.5M盐酸溶液析出固体，过滤，烘干。加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，烘干。After reacting for 1.5 hours, HPLC detected that the reaction was complete, filtered the reaction solution, poured it into a reaction flask, added diethylamine to react for 20 minutes, concentrated to a small amount, added 0.5M hydrochloric acid solution to precipitate a solid, filtered, and dried. Add 500 mL of diethylamine to react for 20 minutes, concentrate to a small amount, add petroleum ether to precipitate a solid, filter, and dry.

化合物6的HPLC检测条件为：The HPLC detection condition of compound 6 is:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000006
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000006

梯度：0-30min，70％B-90％B；Gradient: 0-30min, 70%B-90%B;

收率：80％，纯度：85％。Yield: 80%, purity: 85%.

7.合成化合物7：7. Synthesis of compound 7:

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMeBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe

准确称取Boc-Pyr-OH(74.5mmol)、BOP(74.5mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(74.5mmol)，再将H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe(67.7mmol)用DMF溶解后加入反应中开始反应。Accurately weigh Boc-Pyr-OH (74.5mmol) and BOP (74.5mmol) in a reaction flask, dissolve them completely in DMF, and then add DIEA (74.5mmol) in a cold bath for 10 minutes, then add H-His(Trt)-Trp( Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe (67.7mmol) was dissolved in DMF and added to the reaction to start the reaction.

反应1.5h后HPLC检测反应完全，过滤反应液，滤渣用DMF洗涤两次，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。HPLC检测条件如下：After reacting for 1.5 h, HPLC detected that the reaction was complete, filtered the reaction solution, washed the filter residue twice with DMF, precipitated with 0.5M hydrochloric acid, collected the solid after filtration, washed with purified water until neutral (detected by pH test paper), dried, and weighed. HPLC detection conditions are as follows:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈Mobile phase A: 0.1% TFA/water, Mobile phase B: 0.1% TFA/acetonitrile

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000007
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000007

梯度：0-30min，80％B-95％B；Gradient: 0-30min, 80%B-95%B;

收率：82％，纯度：85.1％。Yield: 82%, purity: 85.1%.

8、合成化合物8：8. Synthesis of compound 8:

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OHBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OH

称取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe(55.5mmol)，置于1110ml甲醇中搅拌5min完全溶解后，取110ml的2MNaOH缓慢加入反应，开始反应。Weigh Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe (55.5mmol), put it in 1110ml methanol and stir for 5min to dissolve completely, then take 110ml of 2M NaOH was slowly added to the reaction to start the reaction.

反应3h后HPLC检测反应完全，加入盐酸溶液，沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重；HPLC检测条件如下：After reacting for 3 hours, HPLC detected that the reaction was complete, added hydrochloric acid solution, precipitated, collected the solid after filtration, washed with purified water until neutral (pH test paper detection), dried, and weighed; HPLC detection conditions are as follows:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000008
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000008

梯度：0-30min，80％B-95％B；Gradient: 0-30min, 80%B-95%B;

收率：78％，纯度：88.9％。Yield: 78%, purity: 88.9%.

9、合成化合物9：H-Arg(pbf)-Pro-NHEt9. Synthesis of Compound 9: H-Arg(pbf)-Pro-NHEt

准确称取Fmoc-Arg(pbf)-OH(150mmol)、BOP(157.5mmol)于反应瓶中，用DMF完全溶解后冷浴10min，加入DIEA，撤掉冷浴，反应20min。Accurately weigh Fmoc-Arg(pbf)-OH (150mmol) and BOP (157.5mmol) in a reaction flask, dissolve them completely in DMF, then cool in a 10min bath, add DIEA, remove the cooling bath, and react for 20min.

称取H-Pro-NHEt.HCl(157.5mmol)于三角瓶中，用DMF完全溶解后加入TEA(157.5mmol)，快速混匀后加入上反应中开始反应。Weigh H-Pro-NHEt.HCl (157.5mmol) in a Erlenmeyer flask, dissolve it completely with DMF, add TEA (157.5mmol), mix quickly and add to the above reaction to start the reaction.

反应2h后HPLC检测反应完全，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥；加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。HPLC检测条件如下：After 2 hours of reaction, HPLC detected that the reaction was complete, precipitated with 0.5M hydrochloric acid, collected the solid after filtration, then washed with purified water until neutral (detected by pH test paper), and dried; added 500 mL of diethylamine to react for 20 minutes, concentrated to a small amount, added petroleum ether to precipitate The solid was filtered and dried in vacuo. HPLC detection conditions are as follows:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000009
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000009

梯度：0-30min，0％B-50％B。Gradient: 0-30min, 0%B-50%B.

收率88％，纯度：78％。Yield: 88%, purity: 78%.

10、合成化合物10：10. Synthesis of compound 10:

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-Arg(pbf)-Pro-NHEtBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-Arg(pbf)-Pro-NHEt

准确称取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OH(43.2mmol)、BOP(45mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(45mmol)，再将H-Arg(pbf)-Pro-NHEt(45mmol)用DMF溶解后加入反应中开始反应。Accurately weigh Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OH(43.2mmol), BOP(45mmol) in the reaction flask, and use DMF After complete dissolution, add DIEA (45mmol) in a cold bath for 10min, then dissolve H-Arg(pbf)-Pro-NHEt (45mmol) in DMF and add to the reaction to start the reaction.

反应1.0h后HPLC检测反应完全，加入0.5M盐酸析出固体，过滤收集固体， 然后用纯化水洗至中性(pH试纸检测)，干燥，称重。其结果参照图11，收率102.1％，纯度：74.9％。After reacting for 1.0 h, HPLC detected that the reaction was complete, and 0.5M hydrochloric acid was added to precipitate a solid, which was collected by filtration, washed with purified water until neutral (detected by pH test paper), dried, and weighed. As a result, referring to FIG. 11 , the yield was 102.1%, and the purity was 74.9%.

HPLC检测条件为：HPLC detection condition is:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000010
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000010

梯度：0-30min，70％B-80％B。Gradient: 0-30min, 70%B-80%B.

11、合成丙氨瑞林粗品：Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt11. Synthesis of crude alanorelin: Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt

将化合物10(43.2mmol)加入反应瓶中，加入300ml裂解液(TFA∶TIS∶H2O＝95∶2.5∶2.5)反应30min后用冷冻乙醚沉淀，过滤，收集固体，得到产物粗品，产物用水溶解后HPLC检测分析。结果参照图1，其收率：86.7％，纯度97.2％。Add compound 10 (43.2mmol) into the reaction flask, add 300ml of lysate (TFA:TIS:H2O=95:2.5:2.5) and react for 30min, then precipitate with frozen ether, filter, collect the solid, and obtain the crude product, which is dissolved in water HPLC detection and analysis. The result is referring to Fig. 1, and its yield: 86.7%, purity 97.2%.

实施例2Example 2

一种全液相法合成丙氨瑞林的方法，包括以下步骤：A method for the synthesis of Amolide by full liquid phase method, comprising the following steps:

1、合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH1. Synthesis of compound 1: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH

其步骤同实施例1。Its step is with embodiment 1.

2、合成化合物2：Boc-D-Ala-Leu-OEt.HCl2. Synthesis of Compound 2: Boc-D-Ala-Leu-OEt.HCl

准确称取Boc-D-Ala-OH(150mmol)、HOSU(165mmol)于反应瓶中，用DMF完全溶解后，再称取H-Leu-OEt.HCl(165mmol)于三角瓶中，用DMF完全溶解后冷浴10min，加入TEA(165mmol)，快速摇匀，加入上述反应瓶中，继续冷浴5min后加入DCC(165mmol)开始反应。反应1h后HPLC检测反应完全。待完全反应后过滤反应液，用0.5M盐酸水溶液析出，过滤的固体，然后用纯化水洗至中性，30℃干燥。收集固体装至三角瓶中，称重。化合物2的HPLC分析条件同实施例1。其收率：102.6％，纯度：92.6％。Accurately weigh Boc-D-Ala-OH (150mmol) and HOSU (165mmol) in a reaction flask, dissolve them completely with DMF, then weigh H-Leu-OEt.HCl (165mmol) in a conical flask, Cool bath for 10 min after dissolving, add TEA (165 mmol), shake quickly, add to the above reaction flask, continue cooling bath for 5 min, then add DCC (165 mmol) to start the reaction. After 1 h of reaction, HPLC detected that the reaction was complete. After the reaction was complete, the reaction solution was filtered and precipitated with 0.5M hydrochloric acid aqueous solution. The filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solids and put them in a Erlenmeyer flask and weigh them. The HPLC analysis conditions of Compound 2 are the same as those in Example 1. Its yield: 102.6%, purity: 92.6%.

3、合成化合物3：H-D-Ala-Leu-OEt3. Synthesis of compound 3: H-D-Ala-Leu-OEt

准确称取150mmol化合物2于反应瓶中，加入500ml的50％TFA/DCM反应20min，其余同实施例1。收率：85.3％，纯度：90.7％。Accurately weigh 150 mmol of compound 2 into a reaction flask, add 500 ml of 50% TFA/DCM to react for 20 min, and the rest are the same as in Example 1. Yield: 85.3%, purity: 90.7%.

4、合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OEt4. Synthesis of compound 4: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OEt

准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH(98mmol)、BOP(107.8mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(107.8mmol)， 再将H-D-Ala-Leu-OMe(196mmol)用DCM溶解后加入反应中开始反应。Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH(98mmol) and BOP(107.8mmol) in the reaction flask, dissolve them completely in DMF, and add DIEA(107.8mmol) in the cooling bath for 10min , and then H-D-Ala-Leu-OMe (196mmol) was dissolved in DCM and added to the reaction to start the reaction.

反应1.0h后HPLC检测反应完全，浓缩，用0.5M盐酸沉淀，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，称重；HPLC检测条件同实施例1。After reacting for 1.0 h, HPLC detected that the reaction was complete, concentrated, precipitated with 0.5M hydrochloric acid, collected the solid by filtration, washed with purified water until neutral (detected by pH test paper), and weighed; the HPLC detection conditions were the same as in Example 1.

其收率：94.5％，纯度：80.3％。Its yield: 94.5%, purity: 80.3%.

5、合成化合物5：H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OEt5. Synthesis of compound 5: H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OEt

其步骤同实施例1。Its step is with embodiment 1.

6、合成化合物6：H-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OEt6. Synthesis of compound 6: H-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OEt

准确称取H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe(169.4mmol)、Fmoc-His(Trt)-OH(93.2mmol)、HOBt(93.2mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DCC(93.2mmol)开始反应。Accurately weigh H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe(169.4mmol), Fmoc-His(Trt)-OH(93.2mmol), HOBt(93.2mmol) In the reaction bottle, after completely dissolving with DMF, add DCC (93.2 mmol) to start the reaction after cooling for 10 min.

反应1.5h后HPLC检测反应完全，过滤反应液，倒入反应瓶中，再加入二乙胺反应20min，浓缩至少量，加入0.5M盐酸溶液析出固体，过滤，烘干。加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，烘干。收率：80.3％，纯度：84.5％。After reacting for 1.5 hours, HPLC detected that the reaction was complete, filtered the reaction solution, poured it into a reaction flask, added diethylamine to react for 20 minutes, concentrated to a small amount, added 0.5M hydrochloric acid solution to precipitate a solid, filtered, and dried. Add 500 mL of diethylamine to react for 20 minutes, concentrate to a small amount, add petroleum ether to precipitate a solid, filter, and dry. Yield: 80.3%, purity: 84.5%.

7.合成化合物7：7. Synthesis of compound 7:

Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OEtFmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OEt

准确称取Fmoc-Pyr-OH(135.4mmol)、BOP(135.4mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(135.4mmol)，再将H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe(67.7mmol)用DMF溶解后加入反应中开始反应。Accurately weigh Fmoc-Pyr-OH (135.4mmol) and BOP (135.4mmol) in the reaction flask, dissolve it completely with DMF, then add DIEA (135.4mmol) in the cooling bath for 10min, then add H-His(Trt)-Trp( Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe (67.7mmol) was dissolved in DMF and added to the reaction to start the reaction.

反应1.5h后HPLC检测反应完全，过滤反应液，滤渣用DMF洗涤两次，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。收率：82％，纯度：85.1％。After reacting for 1.5 h, HPLC detected that the reaction was complete, filtered the reaction solution, washed the filter residue twice with DMF, precipitated with 0.5M hydrochloric acid, collected the solid after filtration, washed with purified water until neutral (detected by pH test paper), dried, and weighed. Yield: 82%, purity: 85.1%.

8、合成化合物8：8. Synthesis of compound 8:

Fmoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OHFmoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OH

称取Fmoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe(55.5mmol)，置于1110ml甲醇中搅拌5min完全溶解后，取1110ml的2MNaOH缓慢加入反应，开始反应。Weigh Fmoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe (55.5mmol), place in 1110ml of methanol and stir for 5min to completely dissolve, then take 1110ml of 2M NaOH was slowly added to the reaction to start the reaction.

反应3h后HPLC检测反应完全，加入盐酸溶液，沉淀，过滤后收集固体， 然后用纯化水洗至中性(pH试纸检测)，干燥，称重；收率：77.8％，纯度：88.5％。After 3 hours of reaction, HPLC detected that the reaction was complete, added hydrochloric acid solution, precipitated, collected the solid after filtration, then washed it with purified water until neutral (detected by pH test paper), dried, and weighed; yield: 77.8%, purity: 88.5%.

9、合成化合物9：H-Arg(pbf)-Pro-NHEt9. Synthesis of Compound 9: H-Arg(pbf)-Pro-NHEt

准确称取Boc-Arg(pbf)-OH(150mmol)、BOP(157.5mmol)于反应瓶中，用DMF完全溶解后冷浴10min，加入DIEA，撤掉冷浴，反应20min。Accurately weigh Boc-Arg(pbf)-OH (150mmol) and BOP (157.5mmol) in a reaction flask, dissolve them completely in DMF, then cool them for 10 minutes, add DIEA, remove the cooling bath, and react for 20 minutes.

称取H-Pro-NHEt.HCl(157.5mmol)于三角瓶中，用DMF完全溶解后加入TEA(157.5mmol)，快速混匀后加入上反应中开始反应。Weigh H-Pro-NHEt.HCl (157.5mmol) in a Erlenmeyer flask, dissolve it completely with DMF, add TEA (157.5mmol), mix quickly and add to the above reaction to start the reaction.

反应2h后HPLC检测反应完全，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥；加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。收率：87.8％，纯度：77.8％。After 2 hours of reaction, HPLC detected that the reaction was complete, precipitated with 0.5M hydrochloric acid, collected the solid after filtration, then washed with purified water until neutral (detected by pH test paper), and dried; added 500 mL of diethylamine to react for 20 minutes, concentrated to a small amount, added petroleum ether to precipitate The solid was filtered and dried in vacuo. Yield: 87.8%, purity: 77.8%.

10、合成化合物10：10. Synthesis of compound 10:

Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-Arg-Pro-NHEtFmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-Arg-Pro-NHEt

准确称取Fmoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OH(43.2mmol)、BOP(45mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(45mmol)，再将H-Arg(pbf)-Pro-NHEt(86.4mmol)用DMF溶解后加入反应中开始反应。Accurately weigh Fmoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OH(43.2mmol), BOP(45mmol) in the reaction flask, and use DMF After complete dissolution, add DIEA (45 mmol) in a cooling bath for 10 min, then dissolve H-Arg(pbf)-Pro-NHEt (86.4 mmol) in DMF and add to the reaction to start the reaction.

反应1.0h后HPLC检测反应完全，加入0.5M盐酸析出固体，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。收率：102％，纯度：74.5％。After 1.0 h of reaction, HPLC detected that the reaction was complete, and 0.5M hydrochloric acid was added to precipitate a solid, which was collected by filtration, washed with purified water until neutral (detected by pH test paper), dried, and weighed. Yield: 102%, purity: 74.5%.

11、合成丙氨瑞林粗品：Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt11. Synthesis of crude alanorelin: Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt

将化合物10(42.1mmol)加入反应瓶中，加入300ml裂解液(TFA∶TIS∶H2O＝95∶2.5∶2.5)反应30min后用冷冻乙醚沉淀，过滤，收集固体，得到产物粗品，产物用水溶解后HPLC检测分析。其收率：86.3％，纯度96.9％。Add compound 10 (42.1 mmol) into the reaction flask, add 300ml of lysate (TFA:TIS:H2O=95:2.5:2.5) and react for 30min, then precipitate with frozen ether, filter, collect the solid, and obtain the crude product, which is dissolved in water HPLC detection and analysis. Its yield: 86.3%, purity 96.9%.

实施例3Example 3

一种全液相法合成地洛瑞林的方法，包括以下步骤：A method for synthesizing deslorelin by a full liquid phase method, comprising the following steps:

1、合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH1. Synthesis of compound 1: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH

将Fmoc-Trp(Boc)-Ser(tBu)-OSu(100mmol)用DMF完全溶解后，再准确称取H-Tyr(tBu)-OH(110mmol)加入上述反应瓶，加TEA(110mmol)开始反应。Dissolve Fmoc-Trp(Boc)-Ser(tBu)-OSu(100mmol) completely in DMF, then accurately weigh H-Tyr(tBu)-OH(110mmol) into the above reaction flask, add TEA(110mmol) to start the reaction .

搅拌反应60min后，HPLC检测反应完全。After stirring and reacting for 60 min, HPLC detected that the reaction was complete.

将反应液分两次倒入三角瓶中，再加入0.5M盐酸快速搅拌析出，过滤的固体，然后用纯化水洗至中性，30℃干燥。收集固体装至容器中，称重。化合物1的HPLC检测条件为：The reaction solution was poured into the Erlenmeyer flask twice, and then 0.5M hydrochloric acid was added to rapidly stir and precipitate, and the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solids into containers and weigh. The HPLC detection condition of compound 1 is:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000011
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000011

梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.

收率：97％；纯度：87.5％。Yield: 97%; Purity: 87.5%.

2、合成化合物2：Fmoc-D-Trp-Leu-OMe.HCl2. Synthesis of compound 2: Fmoc-D-Trp-Leu-OMe.HCl

准确称取Fmoc-D-Trp-OH(150mmol)、HOSU(165mmol)于反应瓶中，用DMF完全溶解后，再称取H-Leu-OMe.HCl(165rmmol)于三角瓶中，用DMF完全溶解后冷浴10min，加入TEA(165mmol)，快速摇匀，加入上述反应瓶中，继续冷浴5min后加入DCC(165mmol)开始反应。反应1.5h后HPLC检测反应完全。Accurately weigh Fmoc-D-Trp-OH (150mmol) and HOSU (165mmol) in a reaction flask, dissolve them completely with DMF, then weigh H-Leu-OMe.HCl (165mmol) in a conical flask, Cool bath for 10 min after dissolving, add TEA (165 mmol), shake quickly, add to the above reaction flask, continue cooling bath for 5 min, then add DCC (165 mmol) to start the reaction. After 1.5 h of reaction, HPLC detected that the reaction was complete.

待完全反应后过滤反应液，用0.5M盐酸水溶液析出，过滤的固体，然后用纯化水洗至中性，30℃干燥。收集固体装至三角瓶中，称重；化合物2的HPLC分析条件为：After the reaction was complete, the reaction solution was filtered and precipitated with 0.5M hydrochloric acid aqueous solution. The filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solid and put it in the Erlenmeyer flask and weigh it; the HPLC analysis conditions of compound 2 are:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000012
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000012

梯度：0～30min，50％B～90％B；Gradient: 0~30min, 50%B~90%B;

收率：103.6％，纯度：94.1％。Yield: 103.6%, purity: 94.1%.

3、合成化合物3：H-D-Trp-Leu-OMe3. Synthesis of compound 3: H-D-Trp-Leu-OMe

准确称取Fmoc-D-Trp-Leu-OMe.HCl(150mmol)于反应瓶中，加入二乙胺400ml反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。化合物3的HPLC检测条件为：Accurately weigh Fmoc-D-Trp-Leu-OMe.HCl (150mmol) into a reaction flask, add 400ml of diethylamine to react for 20min, concentrate to a small amount, add petroleum ether to precipitate a solid, filter, and vacuum dry. The HPLC detection condition of compound 3 is:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000013
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000013

梯度：0～30min，3％B～10％B。Gradient: 0~30min, 3%B~10%B.

收率：89.2％，纯度：89.4％。Yield: 89.2%, purity: 89.4%.

4、合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe4. Synthesis of compound 4: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe

准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH(97mmol)、BOP(106.7 mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(106.7mmol)，再将H-D-Trp-Leu-OMe(106.7mmol)用DCM溶解后加入反应中开始反应。Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH(97mmol) and BOP(106.7mmol) in the reaction flask, dissolve them completely in DMF, and then add DIEA(106.7mmol) in the cooling bath for 10min , and then H-D-Trp-Leu-OMe (106.7mmol) was dissolved in DCM and added to the reaction to start the reaction.

反应1.0h后HPLC检测反应完全，浓缩，用0.5M盐酸沉淀，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，称重；化合物4的HPLC检测条件为：After reacting for 1.0 h, HPLC detected that the reaction was complete, concentrated, precipitated with 0.5M hydrochloric acid, collected the solid by filtration, washed with purified water to neutral (pH test paper detection), and weighed; the HPLC detection conditions of compound 4 were:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000014
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000014

梯度：0-30min，50％B-90％B。Gradient: 0-30min, 50%B-90%B.

收率：92.3％；纯度：81％。Yield: 92.3%; Purity: 81%.

5、合成化合物5：H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe5. Synthesis of Compound 5: H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe

准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe(89.5mmol)于反应瓶中，加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。化合物5的HPLC分析条件为：Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe (89.5mmol) in a reaction bottle, add 500mL diethylamine to react for 20min, concentrate to a certain amount, add petroleum Ether precipitated as a solid, which was filtered and dried in vacuo. The HPLC analysis conditions of compound 5 are:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000015
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000015

梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.

收率：97％；纯度：85.4％。Yield: 97%; Purity: 85.4%.

6、合成化合物6：H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe6. Synthesis of compound 6: H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe

准确称取H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe(86.8mmol)、Fmoc-His(Trt)-OH(95.5mmol)、HOBt(95.5mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DCC(95.5mmol)开始反应。Accurately weigh H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe(86.8mmol), Fmoc-His(Trt)-OH(95.5mmol), HOBt(95.5mmol) In the reaction bottle, after completely dissolving with DMF, add DCC (95.5 mmol) to start the reaction after cooling for 10 min.

反应1.5h后HPLC检测反应完全，过滤反应液，倒入反应瓶中，再加入二乙胺反应20min，浓缩至少量，加入0.5M盐酸溶液析出固体，过滤，烘干。加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，烘干。After reacting for 1.5 hours, HPLC detected that the reaction was complete, filtered the reaction solution, poured it into a reaction flask, added diethylamine to react for 20 minutes, concentrated to a small amount, added 0.5M hydrochloric acid solution to precipitate a solid, filtered, and dried. Add 500 mL of diethylamine to react for 20 minutes, concentrate to a small amount, add petroleum ether to precipitate a solid, filter, and dry.

化合物6的HPLC检测条件为：The HPLC detection condition of compound 6 is:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000016
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000016

梯度：0-30min，70％B-90％B；Gradient: 0-30min, 70%B-90%B;

收率：85％，纯度：81.8％。Yield: 85%, purity: 81.8%.

7.合成化合物7：7. Synthesis of compound 7:

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMeBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe

准确称取Boc-Pyr-OH(81.2mmol)、BOP(81.2mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(81.2mmol)，再将H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe(73.8mmol)用DMF溶解后加入反应中开始反应。Accurately weigh Boc-Pyr-OH (81.2mmol) and BOP (81.2mmol) in a reaction flask, dissolve them completely in DMF, then add DIEA (81.2mmol) in a cold bath for 10min, then add H-His(Trt)-Trp( Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe (73.8mmol) was dissolved in DMF and added to the reaction to start the reaction.

反应1.5h后HPLC检测反应完全，过滤反应液，滤渣用DMF洗涤两次，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。HPLC检测条件如下：After reacting for 1.5 h, HPLC detected that the reaction was complete, filtered the reaction solution, washed the filter residue twice with DMF, precipitated with 0.5M hydrochloric acid, collected the solid after filtration, washed with purified water until neutral (detected by pH test paper), dried, and weighed. HPLC detection conditions are as follows:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈Mobile phase A: 0.1% TFA/water, Mobile phase B: 0.1% TFA/acetonitrile

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000017
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000017

梯度：0-30min，80％B-95％B；Gradient: 0-30min, 80%B-95%B;

收率：81％，纯度：85.1％。Yield: 81%, purity: 85.1%.

8、合成化合物8：8. Synthesis of compound 8:

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OHBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OH

称取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe(59.8mmol)，置于1110ml甲醇中搅拌5min完全溶解后，取110ml的2MNaOH缓慢加入反应，开始反应。Weigh Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe (59.8mmol), put it in 1110ml methanol and stir for 5min to dissolve completely, then take 110ml of 2M NaOH was slowly added to the reaction to start the reaction.

反应3h后HPLC检测反应完全，加入盐酸溶液，沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重；HPLC检测条件如下：After reacting for 3 hours, HPLC detected that the reaction was complete, added hydrochloric acid solution, precipitated, collected the solid after filtration, washed with purified water until neutral (pH test paper detection), dried, and weighed; HPLC detection conditions are as follows:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000018
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000018

梯度：0-30min，70％B-90％B；Gradient: 0-30min, 70%B-90%B;

收率：74％，纯度：88.9％。Yield: 74%, purity: 88.9%.

9、合成化合物9：H-Arg(pbf)-Pro-NHEt9. Synthesis of Compound 9: H-Arg(pbf)-Pro-NHEt

准确称取Fmoc-Arg(pbf)-OH(150mmol)、BOP(157.5mmol)于反应瓶中，用DMF完全溶解后冷浴10min，加入DIEA，撤掉冷浴，反应20min。Accurately weigh Fmoc-Arg(pbf)-OH (150mmol) and BOP (157.5mmol) in a reaction flask, dissolve them completely in DMF, then cool in a 10min bath, add DIEA, remove the cooling bath, and react for 20min.

称取H-Pro-NHEt.HCl(157.5mmol)于三角瓶中，用DMF完全溶解后加入TEA(157.5mmol)，快速混匀后加入上反应中开始反应。Weigh H-Pro-NHEt.HCl (157.5mmol) in a Erlenmeyer flask, dissolve it completely with DMF, add TEA (157.5mmol), mix quickly and add to the above reaction to start the reaction.

反应2h后HPLC检测反应完全，用0.5M盐酸沉淀，过滤后收集固体，然后 用纯化水洗至中性(pH试纸检测)，干燥；加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。HPLC检测条件如下：After 2 hours of reaction, HPLC detected that the reaction was complete, precipitated with 0.5M hydrochloric acid, collected the solid after filtration, then washed with purified water until neutral (detected by pH test paper), and dried; added 500 mL of diethylamine to react for 20 minutes, concentrated to a small amount, added petroleum ether to precipitate The solid was filtered and dried in vacuo. HPLC detection conditions are as follows:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000019
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000019

梯度：0-30min，20％B～50％B。Gradient: 0-30min, 20%B～50%B.

收率：88％，纯度：70％。Yield: 88%, purity: 70%.

10、合成化合物10：10. Synthesis of compound 10:

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-Arg(pbf)-Pro-NHEtBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-Arg(pbf)-Pro-NHEt

准确称取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OH(44.2mmol)、BOP(48.6mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(48.6mmol)，再将H-Arg(pbf)-Pro-NHEt(48.6mmol)用DMF溶解后加入反应中开始反应。Accurately weigh Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OH(44.2mmol), BOP(48.6mmol) in the reaction flask, and use After DMF was completely dissolved, DIEA (48.6 mmol) was added in a cold bath for 10 min, and then H-Arg(pbf)-Pro-NHEt (48.6 mmol) was dissolved in DMF and added to the reaction to start the reaction.

反应1.0h后HPLC检测反应完全，加入0.5M盐酸析出固体，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。HPLC检测条件为：After 1.0 h of reaction, HPLC detected that the reaction was complete, and 0.5M hydrochloric acid was added to precipitate a solid, which was collected by filtration, washed with purified water until neutral (detected by pH test paper), dried, and weighed. HPLC detection conditions are:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000020
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000020

梯度：0-30min，20％B～50％B。Gradient: 0-30min, 20%B～50%B.

收率：102.1％，纯度：74.9％。Yield: 102.1%, purity: 74.9%.

11、合成地洛瑞林粗品：Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-NHEt11. Synthesis of crude deslorelin: Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-NHEt

将化合物10(46.1mmol)加入反应瓶中，加入300ml裂解液(TFA∶TIS∶H2O＝95∶2.5∶2.5)反应30min后用冷冻乙醚沉淀，过滤，收集固体，得到产物粗品，产物用水溶解后HPLC检测分析。结果参照图2，其收率：79.7％，纯度93.1％。Add compound 10 (46.1mmol) into the reaction flask, add 300ml of lysate (TFA:TIS:H2O=95:2.5:2.5) and react for 30min, then precipitate with frozen ether, filter, collect the solid, and obtain the crude product, which is dissolved in water HPLC detection and analysis. Result referring to Fig. 2, its yield: 79.7%, purity 93.1%.

实施例4Example 4

一种全液相法合成亮丙瑞林的方法，包括以下步骤：A method for synthesizing leuprolide by a full liquid phase method, comprising the following steps:

1、合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH1. Synthesis of compound 1: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH

将Fmoc-Trp(Boc)-Ser(tBu)-OSu(100mmol)用DMF完全溶解后，再准确称取H-Tyr(tBu)-OH(110mmol)加入上述反应瓶，加TEA(110mmol)开始反 应。Dissolve Fmoc-Trp(Boc)-Ser(tBu)-OSu(100mmol) completely in DMF, then accurately weigh H-Tyr(tBu)-OH(110mmol) into the above reaction flask, add TEA(110mmol) to start the reaction .

搅拌反应60min后，HPLC检测反应完全。After stirring and reacting for 60 min, HPLC detected that the reaction was complete.

将反应液分两次倒入三角瓶中，再加入0.5M盐酸快速搅拌析出，过滤的固体，然后用纯化水洗至中性，30℃干燥。收集固体装至容器中，称重。化合物1的HPLC检测条件为：The reaction solution was poured into the Erlenmeyer flask twice, and then 0.5M hydrochloric acid was added to rapidly stir and precipitate, and the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solids into containers and weigh. The HPLC detection condition of compound 1 is:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000021
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000021

梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.

收率：96.2％；纯度：87.6％。Yield: 96.2%; Purity: 87.6%.

2、合成化合物2：Fmoc-D-Leu-Leu-OMe.HCl2. Synthesis of compound 2: Fmoc-D-Leu-Leu-OMe.HCl

准确称取Fmoc-D-Leu-OH(150mmol)、HOSU(165mmol)于反应瓶中，用DMF完全溶解后，再称取H-Leu-OMe.HCl(165mmol)于三角瓶中，用DMF完全溶解后冷浴10min，加入TEA(165mmol)，快速摇匀，加入上述反应瓶中，继续冷浴5min后加入DCC(165mmol)开始反应。反应1.5h后HPLC检测反应完全。Accurately weigh Fmoc-D-Leu-OH (150mmol) and HOSU (165mmol) in a reaction flask, dissolve them completely with DMF, then weigh H-Leu-OMe.HCl (165mmol) in a conical flask, Cool bath for 10 min after dissolving, add TEA (165 mmol), shake quickly, add to the above reaction flask, continue cooling bath for 5 min, then add DCC (165 mmol) to start the reaction. After 1.5 h of reaction, HPLC detected that the reaction was complete.

待完全反应后过滤反应液，用0.5M盐酸水溶液析出，过滤的固体，然后用纯化水洗至中性，30℃干燥。收集固体装至三角瓶中，称重；化合物2的HPLC分析条件为：After the reaction was complete, the reaction solution was filtered and precipitated with 0.5M hydrochloric acid aqueous solution. The filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solid and put it in the Erlenmeyer flask and weigh it; the HPLC analysis conditions of compound 2 are:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000022
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000022

梯度：0～30min，50％B～90％B；Gradient: 0~30min, 50%B~90%B;

收率：108.1％，纯度：93.4％。Yield: 108.1%, purity: 93.4%.

3、合成化合物3：H-D-Leu-Leu-OMe3. Synthesis of compound 3: H-D-Leu-Leu-OMe

准确称取Fmoc-D-Leu-Leu-OMe.HCl(150mmol)于反应瓶中，加入二乙胺400ml反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。化合物3的HPLC检测条件为：Accurately weigh Fmoc-D-Leu-Leu-OMe.HCl (150mmol) into a reaction flask, add 400ml of diethylamine to react for 20min, concentrate to a small amount, add petroleum ether to precipitate a solid, filter, and vacuum dry. The HPLC detection condition of compound 3 is:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000023
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000023

梯度：0～30min，3％B～10％B。Gradient: 0~30min, 3%B~10%B.

收率：82.4％，纯度：87.2％。Yield: 82.4%, purity: 87.2%.

4、合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe4. Synthesis of compound 4: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe

准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH(97mmol)、BOP(106.7mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(106.7mmol)，再将H-D-Leu-Leu-OMe(106.7mmol)用DCM溶解后加入反应中开始反应。Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH(97mmol) and BOP(106.7mmol) in the reaction flask, dissolve them completely in DMF, and add DIEA(106.7mmol) in the cooling bath for 10min , and then H-D-Leu-Leu-OMe (106.7mmol) was dissolved in DCM and added to the reaction to start the reaction.

反应1.0h后HPLC检测反应完全，浓缩，用0.5M盐酸沉淀，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，称重；化合物4的HPLC检测条件为：After reacting for 1.0 h, HPLC detected that the reaction was complete, concentrated, precipitated with 0.5M hydrochloric acid, collected the solid by filtration, washed with purified water to neutral (pH test paper detection), and weighed; the HPLC detection conditions of compound 4 were:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000024
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000024

梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.

收率：94.1％，纯度：88.6％。Yield: 94.1%, purity: 88.6%.

5、合成化合物5：H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe5. Synthesis of compound 5: H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe

准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe(89.5mmol)于反应瓶中，加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。化合物5的HPLC分析条件为：Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe (89.5mmol) in a reaction bottle, add 500mL of diethylamine to react for 20min, concentrate to a certain amount, add petroleum Ether precipitated as a solid, which was filtered and dried in vacuo. The HPLC analysis conditions of compound 5 are:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000025
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000025

梯度：0-30min，50％B～95％B。Gradient: 0-30min, 50%B～95%B.

收率：93％，纯度：89％。Yield: 93%, purity: 89%.

6、合成化合物6：H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe6. Synthesis of compound 6: H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe

准确称取H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe(86.8mmol)、Fmoc-His(Trt)-OH(95.5mmol)、HOBt(95.5mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DCC(95.5mmol)开始反应。Accurately weigh H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe(86.8mmol), Fmoc-His(Trt)-OH(95.5mmol), HOBt(95.5mmol) In the reaction bottle, after completely dissolving with DMF, add DCC (95.5 mmol) to start the reaction after cooling for 10 min.

反应1.5h后HPLC检测反应完全，过滤反应液，倒入反应瓶中，再加入二乙胺反应20min，浓缩至少量，加入0.5M盐酸溶液析出固体，过滤，烘干。加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，烘干。After reacting for 1.5 hours, HPLC detected that the reaction was complete, filtered the reaction solution, poured it into a reaction flask, added diethylamine to react for 20 minutes, concentrated to a small amount, added 0.5M hydrochloric acid solution to precipitate a solid, filtered, and dried. Add 500 mL of diethylamine to react for 20 minutes, concentrate to a small amount, add petroleum ether to precipitate a solid, filter, and dry.

化合物6的HPLC检测条件为：The HPLC detection condition of compound 6 is:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000026
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000026

梯度：0-30min，70％B-90％B；Gradient: 0-30min, 70%B-90%B;

收率：82.5％，纯度：88％。Yield: 82.5%, purity: 88%.

7.合成化合物7：7. Synthesis of compound 7:

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMeBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe

准确称取Boc-Pyr-OH(76.3mmol)、BOP(76.3mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(76.3mmol)，再将H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe(69.4mmol)用DMF溶解后加入反应中开始反应。Accurately weigh Boc-Pyr-OH (76.3mmol) and BOP (76.3mmol) in the reaction flask, dissolve them completely in DMF, then add DIEA (76.3mmol) in the cold bath for 10min, then add H-His(Trt)-Trp( Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe (69.4mmol) was dissolved in DMF and added to the reaction to start the reaction.

反应1.5h后HPLC检测反应完全，过滤反应液，滤渣用DMF洗涤两次，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。HPLC检测条件如下：After reacting for 1.5 h, HPLC detected that the reaction was complete, filtered the reaction solution, washed the filter residue twice with DMF, precipitated with 0.5M hydrochloric acid, collected the solid after filtration, washed with purified water until neutral (detected by pH test paper), dried, and weighed. HPLC detection conditions are as follows:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈Mobile phase A: 0.1% TFA/water, Mobile phase B: 0.1% TFA/acetonitrile

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000027
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000027

梯度：0-30min，80％B-95％B；Gradient: 0-30min, 80%B-95%B;

收率：80％，纯度：85.1％。Yield: 80%, purity: 85.1%.

8、合成化合物8：8. Synthesis of compound 8:

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OHBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OH

称取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe(55.5mmol)，置于1110ml甲醇中搅拌5min完全溶解后，取110ml的2MNaOH缓慢加入反应，开始反应。Weigh Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OMe (55.5mmol), put it in 1110ml methanol and stir for 5min to dissolve completely, then take 110ml of 2M NaOH was slowly added to the reaction to start the reaction.

反应3h后HPLC检测反应完全，加入盐酸溶液，沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重；HPLC检测条件如下：After reacting for 3 hours, HPLC detected that the reaction was complete, added hydrochloric acid solution, precipitated, collected the solid after filtration, washed with purified water until neutral (pH test paper detection), dried, and weighed; HPLC detection conditions are as follows:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000028
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000028

梯度：0-30min，70％B-90％B；Gradient: 0-30min, 70%B-90%B;

收率：83％，纯度：84％。Yield: 83%, purity: 84%.

9、合成化合物9：H-Arg(pbf)-Pro-NHEt9. Synthesis of Compound 9: H-Arg(pbf)-Pro-NHEt

准确称取Fmoc-Arg(pbf)-OH(150mmol)、BOP(157.5mmol)于反应瓶中，用DMF完全溶解后冷浴10min，加入DIEA，撤掉冷浴，反应20min。Accurately weigh Fmoc-Arg(pbf)-OH (150mmol) and BOP (157.5mmol) in a reaction flask, dissolve them completely in DMF, then cool in a 10min bath, add DIEA, remove the cooling bath, and react for 20min.

称取H-Pro-NHEt.HCl(157.5mmol)于三角瓶中，用DMF完全溶解后加入 TEA(157.5mmol)，快速混匀后加入上反应中开始反应。Weigh H-Pro-NHEt.HCl (157.5mmol) in a Erlenmeyer flask, dissolve it completely with DMF, add TEA (157.5mmol), mix quickly and add to the above reaction to start the reaction.

反应2h后HPLC检测反应完全，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥；加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。HPLC检测条件如下：After 2 hours of reaction, HPLC detected that the reaction was complete, precipitated with 0.5M hydrochloric acid, collected the solid after filtration, then washed with purified water until neutral (detected by pH test paper), and dried; added 500 mL of diethylamine to react for 20 minutes, concentrated to a small amount, added petroleum ether to precipitate The solid was filtered and dried in vacuo. HPLC detection conditions are as follows:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000029
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000029

梯度：0-30min，20％B～50％B。Gradient: 0-30min, 20%B～50%B.

收率：94％，纯度95.6％。Yield: 94%, purity 95.6%.

10、合成化合物10：10. Synthesis of compound 10:

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-Arg(pbf)-Pro-NHEtBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-Arg(pbf)-Pro-NHEt

准确称取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OH(46.1mmol)、BOP(50.7mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(50.7mmol)，再将H-Arg(pbf)-Pro-NHEt(50.7mmol)用DMF溶解后加入反应中开始反应。Accurately weigh Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-OH (46.1mmol), BOP (50.7mmol) in the reaction flask, and use After DMF was completely dissolved, DIEA (50.7 mmol) was added in a cold bath for 10 min, and then H-Arg(pbf)-Pro-NHEt (50.7 mmol) was dissolved in DMF and added to the reaction to start the reaction.

反应1.0h后HPLC检测反应完全，加入0.5M盐酸析出固体，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。HPLC检测条件为：After 1.0 h of reaction, HPLC detected that the reaction was complete, and 0.5M hydrochloric acid was added to precipitate a solid, which was collected by filtration, washed with purified water until neutral (detected by pH test paper), dried, and weighed. HPLC detection conditions are:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000030
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000030

梯度：0-30min，70％B～90％B。Gradient: 0-30min, 70%B～90%B.

收率：104.2％，纯度：80.5％。Yield: 104.2%, purity: 80.5%.

11、合成亮丙瑞林粗品：Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt11. Synthesis of crude leuprolide: Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt

将化合物10(46.1mmol)加入反应瓶中，加入300ml裂解液(TFA∶TIS∶H 2O＝95∶2.5∶2.5)反应30min后用冷冻乙醚沉淀，过滤，收集固体，得到产物粗品，产物用水溶解后HPLC检测分析。检测条件为： Add compound 10 (46.1mmol) into the reaction flask, add 300ml of lysate (TFA:TIS:H 2 O = 95:2.5:2.5) for 30min, precipitate with frozen ether, filter and collect the solid to obtain the crude product, and the product is water After dissolution, HPLC detection and analysis. The detection conditions are:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000031
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000031

梯度：0～30min，10％B～90％B。结果参照图3，其收率：85.8％，纯度91.2％。Gradient: 0~30min, 10%B~90%B. The result is referring to Fig. 3, and its yield: 85.8%, purity 91.2%.

实施例5Example 5

一种全液相法合成组氨瑞林的方法，包括以下步骤：A method for synthesizing histrelin by a full liquid phase method, comprising the following steps:

1、合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH1. Synthesis of compound 1: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH

将Fmoc-Trp(Boc)-Ser(tBu)-OSu(100mmol)用DMF完全溶解后，再准确称取H-Tyr(tBu)-OH(110mmol)加入上述反应瓶，加TEA(110mmol)开始反应。Dissolve Fmoc-Trp(Boc)-Ser(tBu)-OSu(100mmol) completely in DMF, then accurately weigh H-Tyr(tBu)-OH(110mmol) into the above reaction flask, add TEA(110mmol) to start the reaction .

搅拌反应60min后，HPLC检测反应完全。After stirring and reacting for 60 min, HPLC detected that the reaction was complete.

将反应液分两次倒入三角瓶中，再加入0.5M盐酸快速搅拌析出，过滤的固体，然后用纯化水洗至中性，30℃干燥。收集固体装至容器中，称重。化合物1的HPLC检测条件为：The reaction solution was poured into the Erlenmeyer flask twice, and then 0.5M hydrochloric acid was added to rapidly stir and precipitate, and the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solids into containers and weigh. The HPLC detection condition of compound 1 is:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000032
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000032

梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.

收率：96.2％；纯度：87.6％。Yield: 96.2%; Purity: 87.6%.

2、合成化合物2：Fmoc-D-His(Bzl)-Leu-OMe.HCl2. Synthesis of compound 2: Fmoc-D-His(Bzl)-Leu-OMe.HCl

准确称取Fmoc-D-His(Bzl)-OH(150mmol)、HOSU(165mmol)于反应瓶中，用DMF完全溶解后，再称取H-Leu-OMe.HCl(165mmol)于三角瓶中，用DMF完全溶解后冷浴10min，加入TEA(165mmol)，快速摇匀，加入上述反应瓶中，继续冷浴5min后加入DCC(165mmol)开始反应。反应1.5h后HPLC检测反应完全。Accurately weigh Fmoc-D-His(Bzl)-OH (150mmol) and HOSU (165mmol) in the reaction flask, dissolve it completely with DMF, then weigh H-Leu-OMe.HCl (165mmol) in the Erlenmeyer flask, After completely dissolving with DMF, cool bath for 10 min, add TEA (165 mmol), shake quickly, add to the above reaction flask, continue cold bath for 5 min, then add DCC (165 mmol) to start the reaction. After 1.5 h of reaction, HPLC detected that the reaction was complete.

待完全反应后过滤反应液，用0.5M盐酸水溶液析出，过滤的固体，然后用纯化水洗至中性，30℃干燥。收集固体装至三角瓶中，称重；化合物2的HPLC分析条件为：After the reaction was complete, the reaction solution was filtered and precipitated with 0.5M hydrochloric acid aqueous solution. The filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solid and put it in the Erlenmeyer flask and weigh it; the HPLC analysis conditions of compound 2 are:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000033
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000033

梯度：0-30min，50％B-90％B。Gradient: 0-30min, 50%B-90%B.

收率：108.1％，纯度：93.4％。Yield: 108.1%, purity: 93.4%.

3、合成化合物3：H-D-His(Bzl)-Leu-OMe3. Synthesis of compound 3: H-D-His(Bzl)-Leu-OMe

准确称取Fmoc-D-His(Bzl)-Leu-OMe.HCl(150mmol)于反应瓶中，加入二乙胺400ml反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。 化合物3的HPLC检测条件为：Accurately weigh Fmoc-D-His(Bzl)-Leu-OMe.HCl (150mmol) into a reaction flask, add 400ml of diethylamine to react for 20min, concentrate to a small amount, add petroleum ether to precipitate a solid, filter, and vacuum dry. The HPLC detection condition of compound 3 is:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000034
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000034

梯度：0-30min，10％B-50％B。Gradient: 0-30min, 10%B-50%B.

收率：82.4％，纯度：87.2％。Yield: 82.4%, purity: 87.2%.

4、合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe4. Synthesis of compound 4: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe

准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH(96.2mmol)、BOP(105.8mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(105.8mmol)，再将H-D-His(Bzl)-Leu-OMe(105.8mmol)用DCM溶解后加入反应中开始反应。Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH(96.2mmol) and BOP(105.8mmol) in the reaction flask, dissolve them completely in DMF and add DIEA (105.8mmol ), and then H-D-His(Bzl)-Leu-OMe (105.8mmol) was dissolved in DCM and added to the reaction to start the reaction.

反应1.0h后HPLC检测反应完全，浓缩，用0.5M盐酸沉淀，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，称重；化合物4的HPLC检测条件为：After reacting for 1.0 h, HPLC detected that the reaction was complete, concentrated, precipitated with 0.5M hydrochloric acid, collected the solid by filtration, washed with purified water to neutral (pH test paper detection), and weighed; the HPLC detection conditions of compound 4 were:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000035
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000035

梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.

收率：94.1％，纯度：88.6％。Yield: 94.1%, purity: 88.6%.

5、合成化合物5：H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe5. Synthesis of compound 5: H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe

准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe(90.5mmol)于反应瓶中，加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。化合物5的HPLC分析条件为：Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe (90.5mmol) in a reaction flask, add 500mL of diethylamine to react for 20min, concentrate to a certain amount , Add petroleum ether to precipitate a solid, filter and dry in vacuo. The HPLC analysis conditions of compound 5 are:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000036
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000036

梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.

收率：93％，纯度：89％。Yield: 93%, purity: 89%.

6、合成化合物6：6. Synthesis of compound 6:

H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMeH-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe

准确称取H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe(84.1mmol)、Fmoc-His(Trt)-OH(92.5mmol)、HOBt(92.5mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DCC(92.5mmol)开始反应。Accurately weigh H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe(84.1mmol), Fmoc-His(Trt)-OH(92.5mmol), HOBt( 92.5mmol) in the reaction flask, dissolved completely with DMF, cooled in the bath for 10min, and then added DCC (92.5mmol) to start the reaction.

反应1.5h后HPLC检测反应完全，过滤反应液，倒入反应瓶中，再加入二 乙胺反应20min，浓缩至少量，加入0.5M盐酸溶液析出固体，过滤，烘干。加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，烘干。After reacting for 1.5h, HPLC detected that the reaction was complete, filtered the reaction solution, poured it into a reaction flask, added diethylamine to react for 20min, concentrated to a small amount, added 0.5M hydrochloric acid solution to precipitate a solid, filtered, and dried. Add 500 mL of diethylamine to react for 20 minutes, concentrate to a small amount, add petroleum ether to precipitate a solid, filter, and dry.

化合物6的HPLC检测条件为：The HPLC detection condition of compound 6 is:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000037
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000037

梯度：0-30min，70％B-90％B；Gradient: 0-30min, 70%B-90%B;

收率：89％，纯度：85％。Yield: 89%, purity: 85%.

7.合成化合物7：7. Synthesis of compound 7:

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMeBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe

准确称取Boc-Pyr-OH(76.3mmol)、BOP(76.3mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(76.3mmol)，再将H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe(69.4mmol)用DMF溶解后加入反应中开始反应。Accurately weigh Boc-Pyr-OH (76.3mmol) and BOP (76.3mmol) in the reaction flask, dissolve them completely in DMF, then add DIEA (76.3mmol) in the cold bath for 10min, then add H-His(Trt)-Trp( Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe(69.4mmol) was dissolved in DMF and added to the reaction to start the reaction.

反应1.5h后HPLC检测反应完全，过滤反应液，滤渣用DMF洗涤两次，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。HPLC检测条件如下：After reacting for 1.5 h, HPLC detected that the reaction was complete, filtered the reaction solution, washed the filter residue twice with DMF, precipitated with 0.5M hydrochloric acid, collected the solid after filtration, washed with purified water until neutral (detected by pH test paper), dried, and weighed. HPLC detection conditions are as follows:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈Mobile phase A: 0.1% TFA/water, Mobile phase B: 0.1% TFA/acetonitrile

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000038
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000038

梯度：0-30min，80％B-95％B；Gradient: 0-30min, 80%B-95%B;

收率：80％，纯度：85.1％。Yield: 80%, purity: 85.1%.

8、合成化合物8：8. Synthesis of compound 8:

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OHBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OH

取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe(55.5mmol)置于1110ml甲醇中搅拌5min完全溶解后，取110ml的2MNaOH缓慢加入反应，开始反应。Take Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe(55.5mmol) in 1110ml methanol and stir for 5min to completely dissolve, Take 110ml of 2M NaOH and slowly add to the reaction to start the reaction.

反应3h后HPLC检测反应完全，加入盐酸溶液，沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重；HPLC检测条件如下：After reacting for 3 hours, HPLC detected that the reaction was complete, added hydrochloric acid solution, precipitated, collected the solid after filtration, washed with purified water until neutral (pH test paper detection), dried, and weighed; HPLC detection conditions are as follows:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000039
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000039

梯度：0-30min，80％B-95％B；Gradient: 0-30min, 80%B-95%B;

收率：78％，纯度：88.9％。Yield: 78%, purity: 88.9%.

9、合成化合物9：H-Arg(pbf)-Pro-NHEt9. Synthesis of Compound 9: H-Arg(pbf)-Pro-NHEt

准确称取Fmoc-Arg(pbf)-OH(150mmol)、BOP(157.5mmol)于反应瓶中，用DMF完全溶解后冷浴10min，加入DIEA，撤掉冷浴，反应20min。Accurately weigh Fmoc-Arg(pbf)-OH (150mmol) and BOP (157.5mmol) in a reaction flask, dissolve them completely in DMF, then cool in a 10min bath, add DIEA, remove the cooling bath, and react for 20min.

称取H-Pro-NHEt.HCl(157.5mmol)于三角瓶中，用DMF完全溶解后加入TEA(157.5mmol)，快速混匀后加入上反应中开始反应。Weigh H-Pro-NHEt.HCl (157.5mmol) in a Erlenmeyer flask, dissolve it completely with DMF, add TEA (157.5mmol), mix quickly and add to the above reaction to start the reaction.

反应2h后HPLC检测反应完全，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥；加入二乙胺500mL反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。HPLC检测条件如下：After 2 hours of reaction, HPLC detected that the reaction was complete, precipitated with 0.5M hydrochloric acid, collected the solid after filtration, then washed with purified water until neutral (detected by pH test paper), and dried; added 500 mL of diethylamine to react for 20 minutes, concentrated to a small amount, added petroleum ether to precipitate The solid was filtered and dried in vacuo. HPLC detection conditions are as follows:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000040
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000040

梯度：0-30min，0％B-50％B。Gradient: 0-30min, 0%B-50%B.

收率88％，纯度：78％。Yield: 88%, purity: 78%.

10、合成化合物10：10. Synthesis of compound 10:

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-Arg(pbf)-Pro-NHEtBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-Arg(pbf)-Pro-NHEt

取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OH(46.1mmol)、BOP(50.7mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(50.7mmol)，再将H-Arg(pbf)-Pro-NHEt(50.7mmol)用DMF溶解后加入反应中开始反应。Take Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OH(46.1mmol), BOP(50.7mmol) in the reaction flask, After completely dissolving in DMF, add DIEA (50.7 mmol) in a cooling bath for 10 min, then dissolve H-Arg(pbf)-Pro-NHEt (50.7 mmol) in DMF and add to the reaction to start the reaction.

反应1.0h后HPLC检测反应完全，加入0.5M盐酸析出固体，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。HPLC检测条件为：After 1.0 h of reaction, HPLC detected that the reaction was complete, and 0.5M hydrochloric acid was added to precipitate a solid, which was collected by filtration, washed with purified water until neutral (detected by pH test paper), dried, and weighed. HPLC detection conditions are:

流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，

Figure PCTCN2022115636-appb-000041
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
Figure PCTCN2022115636-appb-000041

梯度：0-30min，70％B-80％B。Gradient: 0-30min, 70%B-80%B.

收率104.2％，纯度：80.5％。Yield: 104.2%, purity: 80.5%.

11、合成组氨瑞林粗品：Pyr-His-Trp-Ser-Tyr-D-His-Leu-Arg-Pro-NHEt11. Synthesis of crude histrelin: Pyr-His-Trp-Ser-Tyr-D-His-Leu-Arg-Pro-NHEt

将化合物10(46.1mmol)加入反应瓶中，加入300ml裂解液(TFA∶TIS∶ H 2O＝95∶2.5∶2.5)反应30min后用冷冻乙醚沉淀，过滤，收集固体，得到产物粗品，产物用水溶解后HPLC检测分析。结果参照图4，其收率：85.8％，纯度86.6％。 Add compound 10 (46.1mmol) into the reaction flask, add 300ml of lysate (TFA:TIS: H2O =95:2.5:2.5) for 30min, precipitate with frozen diethyl ether, filter, collect the solid to obtain the crude product, the product is water After dissolution, HPLC detection and analysis. Referring to Fig. 4 for the results, the yield is 85.8%, and the purity is 86.6%.

显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。Apparently, the above-mentioned embodiments are only examples for clear description, rather than limiting the implementation. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is not necessary and impossible to exhaustively list all the implementation manners here. And the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

Claims (10)

一种全液相合成GRnH九肽酰胺类似物的方法，其特征在于，包括如下步骤：A method for synthesizing GRnH nonapeptide amide analogs in full liquid phase, is characterized in that, comprises the steps: S1、液相合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH；S1. Compound 1 synthesized in liquid phase: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH; S2、液相合成化合物2：R 1-R 5-Leu-OR 2； S2, liquid phase synthesis of compound 2: R 1 -R 5 -Leu-OR 2 ; S3、液相合成化合物3：H-R 5-Leu-OR 2； S3, liquid phase synthesis of compound 3: HR 5 -Leu-OR 2 ; S4、液相合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OR 2； S4, liquid phase synthesis of compound 4: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ; S5、液相合成化合物5：H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OR 2； S5, liquid phase synthesis of compound 5: H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ; S6、液相合成化合物6：H-His(R 3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OR 2； S6, liquid phase synthesis of compound 6: H-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ; S7、液相合成化合物7：S7, liquid phase synthesis of compound 7: R 4-Pyr-His(R 3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OR 2； R 4 -Pyr-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ; S8、液相合成化合物8：S8, liquid phase synthesis of compound 8: R 4-Pyr-His(R 3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OH； R 4 -Pyr-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OH; S9、液相合成化合物9：H-Arg(pbf)-Pro-NHEt；S9, liquid phase synthesis of compound 9: H-Arg(pbf)-Pro-NHEt; S10、液相合成化合物10：S10, liquid phase synthesis of compound 10: R 4-Pyr-His(R 3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-Arg(pbf)-Pro-NHEt； R 4 -Pyr-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-Arg(pbf)-Pro-NHEt; S11、GRnH九肽酰胺类似物粗品的制备；S11, preparation of crude product of GRnH nonapeptide amide analog; 其中，R 1为氨基保护基团，包括Fmoc、Z、Boc中的任意一种；R 2为羧基保护基团，包括甲酯Me，乙酯Et，苄酯Bzl，三苯甲酯Tr中的任意一种；R 3包括Boc或Trt中的任意一种；R 4为氨基保护基团，包括Fmoc、Z、Boc中的任意一种；R 5为D-Ala、D-Leu、D-Trp、D-His中的任意一种。 Wherein, R 1 is an amino protecting group, including any one of Fmoc, Z, Boc; R 2 is a carboxyl protecting group, including methyl ester Me, ethyl ester Et, benzyl ester Bzl, trityl ester Tr Any one; R3 includes any one of Boc or Trt; R4 is an amino protecting group, including any one of Fmoc, Z, Boc; R5 is D-Ala, D-Leu, D-Trp , any one of D-His. 根据权利要求1所述的一种全液相合成GRnH九肽酰胺类似物的方法，其特征在于，步骤S1具体包括以下步骤：A method for full liquid phase synthesis of GRnH nonapeptide amide analogs according to claim 1, wherein step S1 specifically comprises the following steps: 以Fmoc-Trp(Boc)-Ser(tBu)-OSu和H-Tyr(tBu)-OH为反应单元进行缩合反应，在溶剂中反应得到化合物1；所述Fmoc-Trp(Boc)-Ser(tBu)-OSu和所述H-Tyr(tBu)-OH的摩尔比为1∶1.05-2，所述H-Tyr(tBu)-OH与所述有机碱的摩尔比为1∶1，所述溶剂包括DMF、THF、甲醇、乙醇、NMP中的任意一种。Take Fmoc-Trp(Boc)-Ser(tBu)-OSu and H-Tyr(tBu)-OH as reaction units to carry out condensation reaction, react in a solvent to obtain compound 1; said Fmoc-Trp(Boc)-Ser(tBu )-OSu and the H-Tyr(tBu)-OH molar ratio is 1:1.05-2, the H-Tyr(tBu)-OH and the organic base molar ratio is 1:1, the solvent Including any one of DMF, THF, methanol, ethanol, and NMP. 根据权利要求1所述的一种全液相合成GRnH九肽酰胺类似物的方法，其特征在于，步骤S2具体包括以下步骤：A method for full liquid phase synthesis of GRnH nonapeptide amide analogs according to claim 1, wherein step S2 specifically comprises the following steps: 以R 1-R 5-OH、H-Leu-OR 2为反应单元进行缩合反应，R 1-R 5-OH与H-Leu-OR 2的 摩尔比为1∶1.05-2，加入活化剂、有机碱、缩合剂，H-Leu-OR 2与活化剂、缩合剂、有机碱的比为1∶1∶1∶1，反应完全后，过滤、析出、洗涤、干燥，收集固体得化合物2； Condensation reaction is carried out with R 1 -R 5 -OH, H-Leu-OR 2 as the reaction unit, the molar ratio of R 1 -R 5 -OH to H-Leu-OR 2 is 1:1.05-2, adding activator, Organic base, condensing agent, the ratio of H-Leu-OR 2 to activator, condensing agent, and organic base is 1:1:1:1, after the reaction is complete, filter, precipitate, wash, dry, and collect the solid to obtain compound 2; 所述活化剂为多肽合成常用的活化剂，包括HOSu、HOBt、HOAt、HOOBt中的任意一种；所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The activator is a commonly used activator for polypeptide synthesis, including any one of HOSu, HOBt, HOAt, HOOBt; the condensing agent is a commonly used condensing agent for polypeptide synthesis, including DCC, DIC, EDC, BOP, pyBOP, AOP , TBTU, HBTU, HATU; the organic base includes any one of DIEA, TEA, NMM; the solvent includes any one of THF, DCM, DMF, NMP, dioxane. 根据权利要求1所述的一种全液相合成GRnH九肽酰胺类似物的方法，其特征在于，步骤S3具体包括以下步骤：A method for full liquid phase synthesis of GRnH nonapeptide amide analogues according to claim 1, wherein step S3 specifically comprises the following steps: 以步骤S2制得的化合物2为底物，加入脱保护试剂和溶剂，浓缩至少量，析出，过滤，真空干燥得化合物3；Using compound 2 prepared in step S2 as a substrate, adding a deprotection reagent and a solvent, concentrating to a small amount, separating out, filtering, and drying in vacuo to obtain compound 3; 所述脱保护试剂包括三氟乙酸、二乙胺、哌嗪、哌啶中的任意一种；所述溶剂为DMF、甲醇、乙醇、DCM、THF中的任意一种。The deprotection reagent includes any one of trifluoroacetic acid, diethylamine, piperazine, and piperidine; the solvent is any one of DMF, methanol, ethanol, DCM, and THF. 根据权利要求1所述的一种全液相合成GRnH九肽酰胺类似物的方法，其特征在于，步骤S4具体包括以下步骤：A method for full liquid phase synthesis of GRnH nonapeptide amide analogs according to claim 1, wherein step S4 specifically comprises the following steps: 以步骤S1合成的化合物1和步骤S3合成的化合物3为反应单元进行缩合反应，其中化合物1和化合物3的摩尔比为1∶1.05-2，加入有机碱、缩合剂，其中化合物3与有机碱、缩合剂的摩尔比为1∶1∶1，在溶剂中反应完全后，浓缩、过滤、洗涤、干燥，得化合物4；Condensation reaction is carried out with compound 1 synthesized in step S1 and compound 3 synthesized in step S3 as reaction units, wherein the molar ratio of compound 1 and compound 3 is 1: 1.05-2, adding organic base and condensing agent, wherein compound 3 and organic base 1. The molar ratio of the condensing agent is 1:1:1. After the reaction in the solvent is complete, concentrate, filter, wash, and dry to obtain compound 4; 所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM species; the solvent includes any one of THF, DCM, DMF, NMP, and dioxane. 根据权利要求1所述的一种全液相合成GRnH九肽酰胺类似物的方法，其特征在于，步骤S5中具体包括以下步骤：A kind of method for full liquid phase synthesis of GRnH nonapeptide amide analog according to claim 1, is characterized in that, specifically comprises the following steps in step S5: 取化合物4，加入脱保护试剂反应脱去Fmoc基团，浓缩至少量，析出固体，过滤，真空干燥得化合物5；所述脱保护试剂包括二乙胺、哌嗪、哌啶溶液的任意一种。Take compound 4, add a deprotection reagent to react to remove the Fmoc group, concentrate to a certain amount, precipitate a solid, filter, and vacuum-dry to obtain compound 5; the deprotection reagent includes any one of diethylamine, piperazine, and piperidine solutions . 根据权利要求1所述的一种全液相合成GRnH九肽酰胺类似物的方法，其特征在于，步骤S6具体包括以下步骤：A method for full liquid phase synthesis of GRnH nonapeptide amide analogs according to claim 1, wherein step S6 specifically comprises the following steps: 以Fmoc-His(R 3)-OH、步骤S5中合成的化合物5为反应单元进行缩合反应，其中化合物5与Fmoc-His(R 3)-OH的摩尔比为1∶1.05-2，加入活化剂、有机碱、缩合剂，其中Fmoc-His(R 3)-OH与活化剂、缩合剂、有机碱的比为1∶1∶1∶1，在溶剂中反应完全，浓缩、过滤、洗涤、干燥，得化合物6； Condensation reaction is carried out with Fmoc-His(R 3 )-OH and compound 5 synthesized in step S5 as the reaction unit, wherein the molar ratio of compound 5 to Fmoc-His(R 3 )-OH is 1:1.05-2, adding activating agent, organic base, condensing agent, wherein the ratio of Fmoc-His(R 3 )-OH to activator, condensing agent, and organic base is 1:1:1:1, the reaction is complete in the solvent, concentrated, filtered, washed, Dry to obtain compound 6; 所述活化剂为多肽合成常用的活化剂，包括HOSu、HOBt、HOAt、HOOBt中的任意一种；所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The activator is a commonly used activator for polypeptide synthesis, including any one of HOSu, HOBt, HOAt, HOOBt; the condensing agent is a commonly used condensing agent for polypeptide synthesis, including DCC, DIC, EDC, BOP, pyBOP, AOP , TBTU, HBTU, HATU; the organic base includes any one of DIEA, TEA, NMM; the solvent includes any one of THF, DCM, DMF, NMP, dioxane. 根据权利要求1所述的一种全液相合成GRnH九肽酰胺类似物的方法，其特征在于，步骤S7具体包括以下步骤：A method for full liquid phase synthesis of GRnH nonapeptide amide analogs according to claim 1, wherein step S7 specifically comprises the following steps: 以R 4-Pyr-OH和步骤S6合成的化合物6进行缩合反应，其中化合物6与R 4-Pyr-OH的摩尔比为1∶1.05-2；加入有机碱、缩合剂，其中R 4-Pyr-OH与缩合剂、有机碱的摩尔比为1∶1∶1，反应完全后，过滤、洗涤、干燥，得化合物7； Perform condensation reaction with R 4 -Pyr-OH and compound 6 synthesized in step S6, wherein the molar ratio of compound 6 to R 4 -Pyr-OH is 1:1.05-2; add organic base and condensing agent, wherein R 4 -Pyr The molar ratio of -OH to condensing agent and organic base is 1:1:1. After the reaction is complete, filter, wash, and dry to obtain compound 7; 所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM species; the solvent includes any one of THF, DCM, DMF, NMP, and dioxane. 根据权利要求1所述的一种全液相合成GRnH九肽酰胺类似物的方法，其特征在于，步骤S8具体包括以下步骤：A method for full liquid phase synthesis of GRnH nonapeptide amide analogs according to claim 1, wherein step S8 specifically comprises the following steps: 取甲醇和化合物7反应，缓慢加入2M NaOH，反应2-4h，过滤、洗涤、干燥得化合物8；Take methanol and compound 7 to react, slowly add 2M NaOH, react for 2-4h, filter, wash and dry to obtain compound 8; 其中，NaOH和化合物7的摩尔比为1.5∶1-20∶1；Wherein, the molar ratio of NaOH and compound 7 is 1.5:1-20:1; 步骤S9具体包括以下步骤：Step S9 specifically includes the following steps: 以R 1-Arg(pbf)-OH、H-Pro-NHEt.HCl为反应单元进行缩合反应，其中R 1-Arg(pbf)-OH和H-Pro-NHEt.HCl的摩尔比为1∶1.05-2，加入有机碱、缩合剂，其中H-Pro-NHEt.HCl与有机碱、缩合剂的摩尔比为1∶1∶1，在溶剂中反应完全后，析出固体，过滤，干燥，脱保护，浓缩，析出固体，过滤，真空干燥得化合物9； Condensation reaction with R 1 -Arg(pbf)-OH, H-Pro-NHEt.HCl as reaction units, wherein the molar ratio of R 1 -Arg(pbf)-OH and H-Pro-NHEt.HCl is 1:1.05 -2. Add organic base and condensing agent, wherein the molar ratio of H-Pro-NHEt.HCl to organic base and condensing agent is 1:1:1. After complete reaction in the solvent, a solid is precipitated, filtered, dried, and deprotected , concentrated, precipitated solid, filtered, and dried in vacuo to obtain compound 9; 所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的 任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM species; the solvent includes any one of THF, DCM, DMF, NMP, and dioxane. 根据权利要求1所述的一种全液相合成GRnH九肽酰胺类似物的方法，其特征在于，步骤S10具体包括以下步骤：A method for full liquid phase synthesis of GRnH nonapeptide amide analogues according to claim 1, wherein step S10 specifically comprises the following steps: 以化合物8和化合物9为反应单元进行缩合反应，其中化合物8和化合物9的摩尔比为1∶1.05-2，加入缩合剂、有机碱，其中化合物9和缩合剂、有机碱的摩尔比为1∶1∶1，反应完全后，过滤、洗涤、干燥得化合物10；Carry out condensation reaction with compound 8 and compound 9 as the reaction unit, wherein the molar ratio of compound 8 and compound 9 is 1: 1.05-2, add condensing agent, organic base, wherein the molar ratio of compound 9 and condensing agent, organic base is 1 : 1:1, after the reaction is complete, filter, wash, and dry to obtain compound 10; 所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种；The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM A kind; Described solvent comprises any one in THF, DCM, DMF, NMP, dioxane; 步骤S11具体包括以下步骤：Step S11 specifically includes the following steps: 取化合物10于反应器中，加入裂解液反应完成后，用冷冻乙醚沉淀，过滤，收集固体，得到GRnH九肽酰胺类似物粗品；Take compound 10 in the reactor, add the lysate and after the reaction is completed, precipitate with frozen ether, filter, collect the solid, and obtain the crude product of GRnH nonapeptide amide analogue; 所述裂解液的组分，按体积比计，包括：TFA∶TIS∶H 2O＝95∶2.5∶2.5。 The components of the lysate, by volume ratio, include: TFA:TIS:H 2 O=95:2.5:2.5.
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