阅读说明：本技术 一种全液相合成那法瑞林的方法 (Method for synthesizing nafarelin in full liquid phase ) 是由 孙鹏程 唐勇擘 王志锋 杜一雄 郭林 余辅松 于 2021-08-30 设计创作，主要内容包括：本发明提供一种全液相合成那法瑞林的方法,涉及医药技术领域。包括如下步骤：采用液相法合成化合物1Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH和化合物2R-(1)-D-2-Nal-Leu-OR-(2),化合物2经脱保护后与化合物1缩合得到化合物4,化合物4经脱保护再接入R-(4)-Pyr-His(R-(3))-片段得到化合物7,化合物7经皂化后接入-Arg(pbf)-Pro-Gly-NH-(2)片段得到化合物10,化合物10经裂解后得到那法瑞林粗品。本发明提供方法制得的那法瑞林纯度可达80％以上。(The invention provides a method for synthesizing nafarelin in a full liquid phase, and relates to the technical field of medicines. The method comprises the following steps: synthesizing compound 1Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH and compound 2R by liquid phase method 1 ‑D‑2‑Nal‑Leu‑OR 2 Compound 2 is condensed with compound 1 after deprotection to obtain compound 4, and compound 4 is inoculated with R after deprotection 4 ‑Pyr‑His(R 3 ) The fragment is obtained to obtain a compound 7, and the compound 7 is inoculated into-Arg (pbf) -Pro-Gly-NH after saponification 2 The fragment is obtained into a compound 10, and the compound 10 is cracked to obtain a crude nafarelin product. The purity of the nafarelin prepared by the method provided by the invention can reach more than 80%.)

1. A method for synthesizing nafarelin in a full liquid phase is characterized by comprising the following steps:

s1, liquid-phase synthesis of compound 1: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH;

s2, liquid-phase synthesis of compound 2: r₁-D-2-Nal-Leu-OR₂；

S3, liquid phase synthesis of compound 3: H-D-2-Nal-Leu-OR₂；

S4, liquid phase synthesis of compound 4: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OR₂；

S5, liquid phase synthesis of compound 5: H-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OR₂；

S6, liquid phase synthesis of compound 6: H-His (R)₃)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OR₂；

S7, liquid phase synthesis of compound 7: r₄-Pyr-His(R₃)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OR₂；

S8, liquid phase synthesis of compound 8: r₄-Pyr-His(R₃)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OH；

S9, liquid phase synthesis of compound 9: H-Arg (pbf) -Pro-Gly-NH₂；

S10, liquid phase synthesis of compound 10:

R₄-Pyr-His(R₃)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-Arg(pbf)-Pro-Gly-NH₂；

s11, preparing a nafarelin crude product;

wherein R is₁Is an amino protecting group, and comprises any one of Fmoc, Z and Boc; r₂Is a carboxyl protecting group, and comprises any one of methyl ester Me, ethyl ester Et, benzyl ester Bzl and trityl ester Tr; r₃Including any of Boc or Trt; r₄Is an amino protecting group and comprises Fmoc, Z,Boc.

2. The method for synthesizing nafarelin in a whole liquid phase according to claim 1, wherein step S1 specifically comprises the following steps:

carrying out condensation reaction by taking Fmoc-Trp (Boc) -Ser (tBu) -OSu and H-Tyr (tBu) -OH as reaction units, and reacting in a solvent to obtain a compound 1; the molar ratio of Fmoc-Trp (Boc) -Ser (tBu) -OSu to H-Tyr (tBu) -OH is 1: 1.05-2, the molar ratio of H-Tyr (tBu) -OH to the organic base is 1: 1, and the solvent comprises any one of DMF, THF, methanol, ethanol and NMP.

3. The method for synthesizing nafarelin in a whole liquid phase according to claim 1, wherein step S2 specifically comprises the following steps:

with R₁-D-2-Nal-OH、H-Leu-OR₂Carrying out a condensation reaction for the reaction unit, R₁-D-2-Nal-OH and H-Leu-OR₂The molar ratio of the active ingredients is 1: 1.05-2, and an activating agent, an organic base and a condensing agent are added into the mixture, H-Leu-OR₂The ratio of the active agent to the condensing agent to the organic base is 1: 1, after the reaction is completed, the mixture is filtered, separated out, washed and dried, and the solid is collected to obtain a compound 2;

the activator is commonly used for polypeptide synthesis and comprises any one of HOSu, HOBt, HOAt and HOOBt; the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane.

4. The method for synthesizing nafarelin in a whole liquid phase according to claim 1, wherein step S3 specifically comprises the following steps:

taking the compound 2 prepared in the step S2 as a substrate, adding a deprotection reagent and a solvent, concentrating to a small amount, precipitating, filtering, and drying in vacuum to obtain a compound 3;

the deprotection reagent comprises any one of trifluoroacetic acid, diethylamine, piperazine and piperidine; the solvent is any one of DMF, methanol, ethanol, DCM and THF.

5. The method for synthesizing nafarelin in a whole liquid phase according to claim 1, wherein step S4 specifically comprises the following steps:

carrying out condensation reaction by taking the compound 1 synthesized in the step S1 and the compound 3 synthesized in the step S3 as reaction units, wherein the molar ratio of the compound 1 to the compound 3 is 1: 1.05-2, adding an organic base and a condensing agent, wherein the molar ratio of the compound 3 to the organic base to the condensing agent is 1: 1, and after the reaction in a solvent is completed, concentrating, filtering, washing and drying to obtain a compound 4;

the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane.

6. The method for synthesizing nafarelin in a whole liquid phase according to claim 1, wherein step S5 specifically comprises the following steps:

taking a compound 4, adding a deprotection reagent to react and remove Fmoc groups, concentrating to a small amount, separating out a solid, filtering, and drying in vacuum to obtain a compound 5; the deprotection reagent comprises any one of diethylamine, piperazine and piperidine solution.

7. The method for synthesizing nafarelin in a whole liquid phase according to claim 1, wherein step S6 specifically comprises the following steps:

with Fmoc-His (R)₃) -OH, compound 5 synthesized in step S5 as a reaction unit, and carrying out a condensation reaction, wherein compound 5 and Fmoc-His (R)₃) The molar ratio of-OH is 1: 1.05-2, and activating agent, organic base and condensing agent are added, wherein Fmoc-His (R) is₃) The ratio of-OH to the activator, the condensing agent and the organic base is 1: 1,reacting completely in a solvent, concentrating, filtering, washing, drying and deprotecting to obtain a compound 6;

the activator is commonly used for polypeptide synthesis and comprises any one of HOSu, HOBt, HOAt and HOOBt; the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane.

8. The method for synthesizing nafarelin in a whole liquid phase according to claim 1, wherein step S7 specifically comprises the following steps:

with R₄A condensation reaction of-Pyr-OH and Compound 6 synthesized in step S6, wherein Compound 6 is reacted with R₄The molar ratio of Pyr-OH is 1: 1.05-2; adding organic base and condensing agent, wherein R₄The mol ratio of-Pyr-OH to the condensing agent to the organic base is 1: 1, after the reaction is completed, the compound is filtered, washed and dried to obtain a compound 7;

the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane.

9. The method for synthesizing nafarelin in a whole liquid phase according to claim 1, wherein step S8 specifically comprises the following steps:

taking methanol to react with the compound 7, slowly adding 2M NaOH, reacting for 2-4h, filtering, washing and drying to obtain a compound 8;

wherein the molar ratio of NaOH to the compound 7 is 1.5: 1-20: 1;

step S9 specifically includes the following steps:

with R₁-Arg(pbf)-OH、H-Pro-Gly-NH₂Condensation reaction of the reaction units, in which H-Pro-Gly-NH₂And R₁-Arg(pbf) The mol ratio of-OH is 1: 1.05-2, and organic base and condensing agent are added, wherein R is₁The mol ratio of Arg (pbf) -OH to organic alkali to condensing agent is 1: 1, solid is separated out after the reaction in solvent is completed, the solid is filtered, dried, deprotected and concentrated, the solid is separated out, filtered and dried in vacuum to obtain a compound 9;

the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane.

10. The method for synthesizing nafarelin in a whole liquid phase according to claim 1, wherein step S10 specifically comprises the following steps:

carrying out condensation reaction by taking a compound 8 and a compound 9 as reaction units, wherein the molar ratio of the compound 8 to the compound 9 is 1: 1.05-2, adding a condensing agent and an organic base, wherein the molar ratio of the compound 9 to the condensing agent to the organic base is 1: 1, and filtering, washing and drying after the reaction is completed to obtain a compound 10;

the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane;

step S11 specifically includes the following steps:

putting the compound 10 into a reactor, adding a cracking solution, precipitating with frozen ether after the reaction is finished, filtering, and collecting a solid to obtain a crude nafarelin product;

the components of the lysis solution comprise, by volume: TFA, TIS, H₂O＝95∶2.5∶2.5。

Technical Field

The invention relates to the technical field of medicines, in particular to a method for synthesizing nafarelin in a full liquid phase.

Background

Nafarelin is a synthetic nonapeptide analogue of gonadotropin releasing hormone (GnRH), and stimulates the pituitary to release Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) at the initial stage of administration, causing a transient elevation of the ovarian steroid hormones; repeated administration can inhibit the pituitary from releasing LH and FSH, reduce the level of estradiol in blood, and achieve the effect of removing ovary, and the inhibition effect can be used for treating hormone-dependent diseases such as endometriosis.

The synthesis of nafarelin in the prior art is mainly a solid phase method, for example, application No. CN201510524010.X is a method for synthesizing pharmaceutical polypeptide nafarelin by a microwave solid phase synthesis method, and the method has the advantages of high production condition requirement, high cost, low yield and unsuitability for large-scale production. At present, no report on the synthesis of nafarelin by a full liquid phase method exists.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the defects of high cost, more solvents, easy-to-use toxic reagents, high environmental protection pressure and low purity of crude products of the current mainstream solid-phase reaction, thereby providing a method for synthesizing nafarelin in a full liquid phase, and being beneficial to large-scale production.

The invention provides a method for synthesizing nafarelin in a full liquid phase, which comprises the following steps:

s1, liquid-phase synthesis of compound 1: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH;

s2, liquid-phase synthesis of compound 2: r₁-D-2-Nal-Leu-OR₂；

S3, liquid phase synthesis of compound 3: H-D-2-Nal-Leu-OR₂；

S4, liquid phase synthesis of compound 4: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OR₂；

S5, liquid phase synthesis of compound 5: H-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OR₂；

S6, liquid phase synthesis of compound 6: H-His (R)₃)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OR₂；

S7, liquid phase synthesis of compound 7: r₄-Pyr-His(R₃)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OR₂；

S8, liquid phase synthesis of compound 8: r₄-Pyr-His(R₃)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OH；

S9, liquid phase synthesis of compound 9: H-Arg (pbf) -Pro-Gly-NH₂；

S10, liquid phase synthesis of compound 10:

R₄-Pyr-His(R₃)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-Arg(pbf)-Pro-Gly-NH₂；

s11, preparing a nafarelin crude product;

wherein R is₁Is an amino protecting group, and comprises any one of Fmoc, Z and Boc; r₂Is a carboxyl protecting group, and comprises any one of methyl ester Me, ethyl ester Et, benzyl ester Bzl and trityl ester Tr; r₃Including any of Boc or Trt; r₄The amino protecting group comprises any one of Fmoc, Z and Boc.

Preferably, step S1 specifically includes the following steps:

carrying out condensation reaction by taking Fmoc-Trp (Boc) -Ser (tBu) -OSu and H-Tyr (tBu) -OH as reaction units, and reacting in a solvent to obtain a compound 1; the molar ratio of Fmoc-Trp (Boc) -Ser (tBu) -OSu to H-Tyr (tBu) -OH is 1: 1.05-2, the molar ratio of H-Tyr (tBu) -OH to the organic base is 1: 1, and the solvent comprises any one of DMF, THF, methanol, ethanol and NMP.

More preferably, the organic base is TEA, the molar ratio of Fmoc-Trp (Boc) -Ser (tBu) -OSu to H-Tyr (tBu) -OH is 1: 1.1, the molar ratio of H-Tyr (tBu) -OH to the organic base is 1: 1, the solvent comprises any one of DMF, THF, methanol, ethanol and NMP, and preferably, the solvent is DMF.

Preferably, step S2 specifically includes the following steps:

with R₁-D-2-Nal-OH、H-Leu-OR₂Is reversedBy condensation reaction of the units R₁-D-2-Nal-OH and H-Leu-OR₂The molar ratio of the active ingredients is 1: 1.05-2, and an activating agent, an organic base and a condensing agent are added into the mixture, H-Leu-OR₂The ratio of the active agent to the condensing agent to the organic base is 1: 1, after the reaction is completed, the mixture is filtered, separated out, washed and dried, and the solid is collected to obtain a compound 2;

the activator is an activator commonly used for polypeptide synthesis, and comprises any one of HOSu, HOBt, HOAt and HOOBt, and more preferably, the activator is HOSu; the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU, and more preferably, the condensing agent is DCC; the organic base comprises any one of DIEA, TEA and NMM, and more preferably, the organic base is TEA; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane, and more preferably the solvent is DMF.

Preferably, step S3 specifically includes the following steps:

taking the compound 2 prepared in the step S2 as a substrate, adding a deprotection reagent and a solvent, concentrating to a small amount, precipitating, filtering, and drying in vacuum to obtain a compound 3;

the deprotection reagent comprises any one of trifluoroacetic acid, diethylamine, piperazine and piperidine; the solvent is any one of DMF, methanol, ethanol, DCM and THF.

Further, R₁The amino protecting agent is preferably Fmoc or Boc.

Further, R₂Is a carboxyl protecting agent, preferably Me or Et; more preferably Me.

When R is₁In Fmoc, the deprotection reagent in step S3 is preferably diethylamine; when R is₁In the case of Boc, the deprotecting reagent is preferably TFA.

Preferably, step S4 specifically includes the following steps:

carrying out condensation reaction by taking the compound 1 synthesized in the step S1 and the compound 3 synthesized in the step S3 as reaction units, wherein the molar ratio of the compound 1 to the compound 3 is 1: 1.05-2, adding an organic base and a condensing agent, wherein the molar ratio of the compound 3 to the organic base to the condensing agent is 1: 1, and after the reaction in a solvent is completed, concentrating, filtering, washing and drying to obtain a compound 4;

the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane. More preferably, the solvent is DMF.

Preferably, step S5 specifically includes the following steps:

taking a compound 4, adding a deprotection reagent to react and remove Fmoc groups, concentrating to a small amount, separating out a solid, filtering, and drying in vacuum to obtain a compound 5; the deprotection reagent comprises any one of diethylamine, piperazine and piperidine solution.

Preferably, step S6 specifically includes the following steps:

with Fmoc-His (R)₃) -OH, compound 5 synthesized in step S5 as a reaction unit, and carrying out a condensation reaction, wherein compound 5 and Fmoc-His (R)₃) The molar ratio of-OH is 1: 1.05-2, and activating agent, organic base and condensing agent are added, wherein Fmoc-His (R) is₃) The ratio of-OH to an activating agent, a condensing agent and organic base is 1: 1, the reaction is completed in a solvent, and a compound 6 is obtained after concentration, filtration, washing, drying and deprotection;

the activator is commonly used for polypeptide synthesis and comprises any one of HOSu, HOBt, HOAt and HOOBt; the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane. More preferably, the solvent is DMF.

Preferably, step S7 specifically includes the following steps:

with R₄A condensation reaction of-Pyr-OH and Compound 6 synthesized in step S6, wherein Compound 6 is reacted with R₄The molar ratio of Pyr-OH is 1: 1.05-2; adding an organic baseA condensing agent, wherein R₄The mol ratio of-Pyr-OH to the condensing agent to the organic base is 1: 1, after the reaction is completed, the compound is filtered, washed and dried to obtain a compound 7;

the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane. More preferably, the solvent is DMF.

Preferably, step S8 specifically includes the following steps:

taking methanol to react with the compound 7, slowly adding 2M NaOH, reacting for 2-4h, filtering, washing and drying to obtain a compound 8;

wherein the molar ratio of NaOH to the compound 7 is 1.5: 1-20: 1.

Preferably, step S9 specifically includes the following steps:

with R₁-Arg(pbf)-OH、H-Pro-Gly-NH₂Condensation reaction of the reaction units, in which H-Pro-Gly-NH₂And R₁The mol ratio of Arg (pbf) -OH is 1: 1.05-2, and organic base and condensing agent are added, wherein R is₁The mol ratio of Arg (pbf) -OH to organic alkali to condensing agent is 1: 1, solid is separated out after the reaction in solvent is completed, the solid is filtered, dried, deprotected and concentrated, the solid is separated out, filtered and dried in vacuum to obtain a compound 9;

the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane. More preferably, the solvent is DMF.

When R is in the specification₁R in-Arg (pbf) -OH₁For the Boc group, 50% TFA/DCM was used as the deprotecting reagent, and pbf group was absent in compounds 9 and 10.

Preferably, step S10 specifically includes the following steps:

carrying out condensation reaction by taking a compound 8 and a compound 9 as reaction units, wherein the molar ratio of the compound 8 to the compound 9 is 1: 1.05-2, adding a condensing agent and an organic base, wherein the molar ratio of the compound 9 to the condensing agent to the organic base is 1: 1, and filtering, washing and drying after the reaction is completed to obtain a compound 10;

the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane. More preferably, the solvent is DMF.

Preferably, step S11 specifically includes the following steps:

putting the compound 10 into a reactor, adding a cracking solution, precipitating with frozen ether after the reaction is finished, filtering, and collecting a solid to obtain a crude nafarelin product;

the components of the lysis solution comprise, by volume: TFA, TIS, H₂O＝95∶2.5∶2.5。

The reagents used in the technical scheme are all common commercially available reagents; in the above technical solution, the precipitation operation usually employs an ether reagent, including any one or any combination of petroleum ether, isopropyl ether and diethyl ether, preferably petroleum ether.

The technical scheme of the invention has the following advantages:

the invention creatively invents a green and mild production process by a full liquid phase synthesis method, does not use any highly toxic and easily toxic reagent, has the purity of over 80 percent, greatly reduces the cost, and is very suitable for large-scale production.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an HPLC chromatogram of a crude synthetic nafarelin of example 1 of the present invention.

Detailed Description

The Chinese names corresponding to the English abbreviations of the substances appearing in the claims and the specification of the present invention are shown in Table 1.

TABLE 1

In the following examples, the purity of each compound and product was analyzed by HPLC, and unless otherwise specified, the HPLC conditions were as follows:

mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;

detection wavelength: 210 nm; flow rate: 1 ml/min; stationary phase: c18 column, 5 μ,

example 1

1. Liquid phase synthesis of compound 1: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH

1.1 feeding:

the materials were dosed as per table 2.

TABLE 2

Abbreviations	Dosage of
		Fmoc-Trp(Boc)-Ser(tBu)-OSU	100mmol
H-Tyr(tBu)-OH	110mmol
		TEA	110mmol
DMF	400ml
		0.5M hydrochloric acid solution	1L

1.2 procedure

After Fmoc-Trp (Boc) -Ser (tBu) -OSu was completely dissolved in DMF, H-Tyr (tBu) -OH was accurately weighed and added to the reaction flask, and TEA (110 mmol) was added to start the reaction. After stirring the reaction for 60min, the reaction was complete as detected by HPLC.

Pouring the reaction solution into a triangular flask twice, adding 0.5M hydrochloric acid, quickly stirring to separate out, filtering to obtain a solid, washing with purified water to be neutral, and drying at 30 ℃. Collecting the solid, loading into a container, and weighing;

yield: 96%, purity: 87.6 percent.

2. Liquid phase synthesis of compound 2: Fmoc-D-2-Nal-Leu-OMe

2.1 charging

The materials were dosed as per table 3.

TABLE 3

Abbreviations	Dosage of
		Fmoc-D-2-Nal-OH	150mmol
HOSU	165mmol
		TEA	165mmol
DCC	165mmol
		H-Leu-OMe.HCl	165mmol
DMF	500ml
		0.5M hydrochloric acid solution	1L

2.2, operation process:

accurately weighing Fmoc-D-2-Nal-OH and HOSU in a reaction bottle, completely dissolving with DMF, then weighing H-Leu-OMe.HCl in a triangular flask, completely dissolving with DMF, cold bathing for 10min, adding TEA, shaking up quickly, adding into the reaction bottle, continuing to cold bath for 5min, adding DCC, and starting reaction.

The reaction was complete after 1.5h by HPLC.

After complete reaction, the reaction solution was filtered, precipitated with 0.5M aqueous hydrochloric acid, and the filtered solid was washed with purified water to neutrality and dried at 30 ℃. Collecting the solid, filling into a triangular flask, and weighing;

yield: 108.1%, purity: 95.7 percent.

3. Liquid phase synthesis of compound 3: H-D-2-Nal-Leu-OMe

3.1 feeding:

the materials were dosed as per table 4.

TABLE 4

Abbreviations	Dosage of
		Fmoc-D-2-Nal-Leu-OMe.HCl	150mmol
Diethylamine	400ml
		Petroleum ether	1L

3.2 procedure

Accurately weighing Fmoc-D-2-Nal-Leu-OMe.HCl in a reaction bottle, adding diethylamine to react for 20min, concentrating to a small amount, adding petroleum ether to precipitate a solid, filtering, and vacuum drying.

Yield: 87.2%, purity: 91 percent.

4. Liquid phase synthesis of compound 4: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OMe

4.1 feeding:

the materials were dosed as per table 5.

TABLE 5

Abbreviations	Dosage of
		Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH	96mmol
BOP	105.6mmol
		TEA	105.6mmol
H-D-2-Nal-Leu-OMe	105.6mmol
		DMF	200ml
DCM	200ml
		0.5M hydrochloric acid solution	1L

4.2 procedure

Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH and BOP are accurately weighed in a reaction bottle, are completely dissolved by DMF, are cooled for 10min, are added with DIEA, and are dissolved by DCM, and then are added into the reaction for starting the reaction.

After the reaction is carried out for 1.0h, HPLC (high performance liquid chromatography) detection shows that the reaction is complete, concentration is carried out, 0.5M hydrochloric acid is used for precipitation, solid is collected by filtration, then purified water is used for washing until the solution is neutral (pH test paper detection), and weighing is carried out;

yield: 92%, purity: 80 percent.

5. Liquid phase synthesis of compound 5: H-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OMe

5.1 charging

The materials were dosed as per table 6.

TABLE 6

Abbreviations	Dosage of
		Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OMe	88.3mmol
Diethylamine	500ml
		Petroleum ether	1L

5.2 procedure

Accurately weighing Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OMe in a reaction bottle, adding diethylamine to react for 20min, concentrating to a small amount, adding petroleum ether to precipitate a solid, filtering, and vacuum drying.

Yield: 92.1%, purity: 85.4 percent

6. Liquid phase synthesis of compound 6: H-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OMe

6.1 charging

The materials were dosed as per table 7.

TABLE 7

6.2 procedure

Fmoc-His (Trt) -OH, HOBt, H-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OMe are accurately weighed into a reaction bottle, and after being completely dissolved by DMF, the reaction bottle is cooled for 10min, and DCC is added to start reaction.

After reacting for 1.5h, detecting by HPLC to complete the reaction, filtering the reaction solution, pouring into a reaction bottle, adding diethylamine for reacting for 20min, concentrating to a small amount, adding 0.5M hydrochloric acid solution to precipitate a solid, filtering, and drying.

Putting Fmoc-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OMe into a reaction bottle, adding diethylamine for deprotection reaction for 20min, concentrating to a small amount, adding petroleum ether to precipitate a solid, filtering and drying.

Yield: 86%, purity: 81.8 percent.

7. Liquid phase synthesis of compound 7: Boc-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OMe

7.1 charging

The materials were dosed as per table 8.

TABLE 8

Abbreviations	Dosage of
		Boc-Pyr-OH	76.9mmol
BOP	76.9mmol
		TEA	76.9mmol
H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe	69.9mmol
		DMF	350ml
0.5M hydrochloric acid solution	1L

7.2 procedure

Accurately weighing Boc-Pyr-OH and BOP into a reaction bottle, completely dissolving the Boc-Pyr-OH and the BOP with DMF, cooling the reaction bottle for 10min, adding DIEA, dissolving H-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OMe with DMF, and adding the solution into the reaction to start the reaction.

After the reaction is carried out for 1.5h, HPLC detection shows that the reaction is complete, the reaction solution is filtered, filter residue is washed twice by DMF, and is precipitated by 0.5M hydrochloric acid, a solid is collected after filtration, and then the solid is washed by purified water to be neutral (pH test paper detection), dried and weighed;

yield: 80%, purity: 85.1 percent.

8. Liquid phase synthesis of compound 8: Boc-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OH

8.1 charging

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe：56mmol

Methanol: 1110ml

2M NaOH：110ml

0.1M hydrochloric acid solution; 2L of

8.2 procedure

Boc-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OMe is taken and put in methanol to be stirred for 5min and completely dissolved, and then 2M NaOH is taken to be slowly added into the mixture for reaction to start the reaction.

After reacting for 3h, detecting the reaction is complete by HPLC, adding a hydrochloric acid solution, precipitating, filtering, collecting a solid, washing with purified water to be neutral (detecting by a pH test paper), drying, and weighing;

yield: 72%, purity: 88.9 percent.

9. Liquid phase synthesis of compound 9: H-Arg (pbf) -Pro-Gly-NH₂

9.1 charging

The materials were dosed as per table 9.

TABLE 9

Abbreviations	Dosage of
		H-Pro-Gly-NH2	150mmol
BOP	157.5mmol
		TEA	157.5mmol
Fmoc-Arg(pbf)-OH	157.5mmol
		TEA	157.5mmol
DMF	400ml
		0.5M hydrochloric acid solution	1L
Diethylamine	500ml

9.2 procedure

Fmoc-Arg (pbf) -OH and BOP are accurately weighed in a reaction flask, and are cooled for 10min after being completely dissolved by DMF, DIEA is added, the cooling bath is removed, and the reaction is carried out for 20 min.

Weighing H-Pro-Gly-NH₂And (3) completely dissolving the mixture in DMF (dimethyl formamide), adding TEA, quickly mixing the mixture uniformly, and adding the mixture into the mixture for reaction to start reaction. After reacting for 2h, precipitating with 0.5M hydrochloric acid, filtering, collecting solid, washing with purified water to neutrality (pH test paper detection), drying, and weighing Fmoc-Arg (pbf) -Pro-Gly-NH₂Adding diethylamine into a reaction bottle, reacting for 20min, concentrating to a small amount, adding petroleum ether to precipitate solid, filtering, and vacuum drying.

Yield 91%, purity: 95.6 percent.

10. Liquid phase synthesis of compound 10:

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-Arg(pbf)-Pro-Gly-NH₂

10.1 charging

The materials were dosed as in table 10.

Watch 10

Abbreviations	Dosage of
		Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OH	40.3mmol
BOP	44.3mmol
		TEA	44.3mmol
H-Arg(pbf)-Pro-Gly-NH2	44.3mmol
		DMF	200ml
0.5M hydrochloric acid solution	500ml

10.2 procedure

Accurately weighing Boc-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OH and BOP into a reaction bottle, completely dissolving with DMF, cooling for 10min, adding DIEA, and adding H-Arg (pbf) -Pro-Gly-NH₂The reaction was started by dissolving it in DMF and adding it to the reaction.

After 1.0h of reaction, HPLC detects the reaction is complete, 0.5M hydrochloric acid is added to precipitate a solid, the solid is collected by filtration, and then the solid is washed to be neutral by purified water (detected by pH test paper), dried and weighed.

Yield 95%, purity: 74.9 percent.

11 Synthesis of crude Nafarelin

11.1 charging

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-Arg(pbf)-Pro-Gly-NH₂：42.1mmol

Lysis buffer (TFA: TIS: H2O ═ 95: 2.5): 300ml

11.2 procedure

Boc-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-Arg (pbf) -Pro-Gly-NH₂Adding the mixture into a reaction bottle, adding a lysis solution, reacting for 30min, precipitating with frozen ether, filtering, collecting solid to obtain a crude product, dissolving the product with water, and detecting and analyzing by HPLC.

Yield: 86% and purity 83.5%.

Example 2

A method for synthesizing nafarelin by a full liquid phase method comprises the following steps:

1. liquid phase synthesis of compound 1: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH

The procedure is as in example 1.

2. Liquid phase synthesis of compound 2: Boc-D-2-Nal-Leu-OEt. HCl

2.1 charging

The materials were dosed as in table 11.

TABLE 11

Material(s)	Dosage of
		Boc-D-2-Nal-OH	150mmol
HOSU	165mmol
		TEA	165mmol
DCC	165mmol
		H-Leu-OEt.HCl	300mmol
DMF	500ml
		0.5M hydrochloric acid solution	1L

2.2, operation process:

accurately weighing Boc-D-2-Nal-OH and HOSU in a reaction bottle, completely dissolving with DMF, weighing H-Leu-OEt. HCl in a triangular flask, completely dissolving with DMF, cold-bathing for 10min, adding TEA, shaking up quickly, adding into the reaction bottle, continuing cold-bathing for 5min, adding DCC, and starting reaction. After 1h the reaction was complete as determined by HPLC. After complete reaction, the reaction solution was filtered, precipitated with 0.5M aqueous hydrochloric acid, and the filtered solid was washed with purified water to neutrality and dried at 30 ℃. The solid was collected, filled into a flask, and weighed. Yield: 107.6%, purity: 95.6 percent.

3. Liquid phase synthesis of compound 3: H-D-2-Nal-Leu-OEt

Boc-D-2-Nal-Leu-OMe. HCl was weighed out accurately into a reaction flask, 500ml 50% TFA/DCM was added for 20min, concentrated to a small amount, ether was added to precipitate a solid, filtered and dried in vacuo. The rest is the same as example 1.

Yield: 93% and the purity is 96.6%.

4. Liquid phase synthesis of compound 4: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OEt

4.1 feeding:

the amounts charged are shown in Table 12.

TABLE 12

Material(s)	Dosage of
		Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH	98mmol
BOP	107.8mmol
		TEA	107.8mmol
H-D-2-Nal-Leu-OEt	196mmol
		DMF	200ml
DCM	200ml
		0.5M hydrochloric acid solution	1L

The procedure was as in example 1.

The yield thereof is as follows: 91.5%, purity: 80.3 percent.

5. Liquid phase synthesis of compound 5: H-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OEt

The procedure is as in example 1.

6. Liquid phase synthesis of compound 6: H-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-2-Nal-Leu-OEt

6.1 charging

The materials were dosed as in table 13.

Watch 13

Material(s)	Dosage of
		Fmoc-His(Boc)-OH	169.4mmol
HOBt	93.2mmol
		DCC	93.2mmol
H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OEt	84.7mmol
		Diethylamine	500ml
DMF	400ml
		0.5M hydrochloric acid solution	1L

The procedure was as in example 1. Yield: 85.3%, purity: 81.5 percent.

7. Liquid phase synthesis of compound 7:

Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OEt

7.1 charging

The batch was carried out according to the contents of Table 14.

TABLE 14

Material(s)	Dosage of
		Fmoc-Pyr-OH	135.4mmol
BOP	74.5mmol
		TEA	74.5mmol
H-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OEt	67.7mmol
		DMF	350ml
0.5M hydrochloric acid solution	1L

The procedure was as in example 1.

Yield: 80.1%, purity: 85 percent.

8. Liquid phase synthesis of compound 8:

Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OH

8.1 charging

Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OEt：55.5mmol

Methanol: 1110ml

2M NaOH：1110ml

0.1M hydrochloric acid solution: 2L of

The procedure was as in example 1.

Yield: 71.8%, purity: 88.5 percent.

9. Liquid phase synthesis of compound 9: H-Arg-Pro-Gly-NH₂

9.1 charging

The amounts charged are shown in Table 15.

Watch 15

Material(s)	Dosage of
		Boc-Arg(pbf)-OH	150mmol
BOP	157.5mmol
		TEA	157.5mmol
H-Pro-Gly-NH2	300mmol
		TEA	157.5mmol
DMF	400ml
		0.5M hydrochloric acid solution	1L
50％TFA/DCM	500ml

The procedure was as in example 1.

Yield: 90.8%, purity: 95.6 percent.

10. Liquid phase synthesis of compound 10:

Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-Arg-Pro-NHEt

10.1 charging

The amounts charged are shown in Table 16.

TABLE 16

Material(s)	Dosage of
		Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OH	43.2mmol
BOP	45mmol
		TEA	45mmol
H-Arg(pbf)-Pro-Gly-NH2	86.4mmol
		DMF	200ml
0.5M hydrochloric acid solution	500ml

The procedure was as in example 1.

Yield 94%, purity: 74.5 percent.

11. Synthesis of crude Nafarelin

Compound 10: 42.1mmol

Lysis solution (TFA: TIS: H)₂O＝95∶2.5∶2.5)：300ml。

The procedure was as in example 1. The yield thereof is as follows: 86.3 percent and the purity of 81.9 percent.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.