Method for synthesizing gonadorelin in full liquid phase

文档序号：1793226 发布日期：2021-11-05 浏览：34次中文

阅读说明：本技术 一种全液相合成戈那瑞林的方法 (Method for synthesizing gonadorelin in full liquid phase ) 是由孙鹏程唐勇擘余辅松杜一雄王志锋郭林于 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戈那瑞林的方法,涉及医药技术领域。S1、液相合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH；S2、液相合成化合物2：R-(1)-Gly-Leu-OR-(2)；S3、液相合成化合物3：H-Gly-Leu-OR-(2)；S4、液相合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OR-(2)；S5、液相合成化合物5：H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OR-(2)；S6、液相合成化合物6：H-His(R-(3))-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OR-(2)；S7、液相合成化合物7：R-(4)-Pyr-His(R-(3))-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OR-(2)；S8、液相合成化合物8：R-(4)-Pyr-His(R-(3))-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OH；S9、液相合成化合物9：H-Arg(pbf)-Pro-Gly-NH-(2)；S10、液相合成化合物10：R-(4)-Pyr-His(R-(3))-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-Arg(pbf)-Pro-Gly-NH-(2)；S11、戈那瑞林粗品的制备。本发明提供的全液相法所制备的戈那瑞林纯度在95％以上。(The invention provides a method for synthesizing gonadorelin in a full liquid phase, and relates to the technical field of medicines. S1, liquid-phase synthesis of compound 1: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH; s2, liquid-phase synthesis of compound 2: r 1 ‑Gly‑Leu‑OR 2 (ii) a S3, liquid phase synthesis of compound 3: H-Gly-Leu-OR 2 (ii) a S4, liquid phase synthesis of compound 4: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-OR 2 (ii) a S5, liquid phase synthesis of compound 5: H-Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-OR 2 (ii) a S6, liquid phase synthesis of compound 6: H-His (R) 3 )‑Trp(Boc)‑Ser(tBu)‑Tyr(tBu)‑Gly‑Leu‑OR 2 (ii) a S7, liquid phase synthesis of compound 7: r 4 ‑Pyr‑His(R 3 )‑Trp(Boc)‑Ser(tBu)‑Tyr(tBu)‑Gly‑Leu‑OR 2 (ii) a S8, liquid phase synthesis of compound 8: r 4 ‑Pyr‑His(R 3 ) -trp (boc) -ser (tbu) -tyr (tbu) -Gly-Leu-OH; s9, liquid phase synthesis of compound 9: H-Arg (pbf) -Pro-Gly-NH 2 (ii) a S10, liquid phase synthesis of compound 10: r 4 ‑Pyr‑His(R 3 )‑Trp(Boc)‑Ser(tBu)‑Tyr(tBu)‑Gly‑Leu‑Arg(pbf)‑Pro‑Gly‑NH 2 (ii) a S11, and preparing a gonadorelin crude product. The purity of the gonadorelin prepared by the full liquid phase method provided by the invention is more than 95%.)

1. A method for synthesizing gonadorelin 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-Gly-Leu-OR₂；

S3, liquid phase synthesis of compound 3: H-Gly-Leu-OR₂；

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

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

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

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

S8, liquid phase synthesis of compound 8: r₄-Pyr-His(R₃)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-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)-Gly-Leu-Arg(pbf)-Pro-Gly-NH₂；

s11, preparing a gonadorelin 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.

2. The method for synthesizing gonadorelin in full liquid phase according to claim 1, wherein the 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 gonadorelin in full liquid phase according to claim 1, wherein the step S2 specifically comprises the following steps:

with R₁-Gly-OH、H-Leu-OR₂Carrying out a condensation reaction for the reaction unit, R₁-Gly-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 gonadorelin in full liquid phase according to claim 1, wherein the 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 gonadorelin in full liquid phase according to claim 1, wherein the 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 gonadorelin in full liquid phase according to claim 1, wherein the 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 gonadorelin in full liquid phase according to claim 1, wherein the 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 an activating agent, a condensing agent and organic base is 1: 1, the reaction is completed in a solvent, and the compound 6 is obtained by concentration, filtration, washing, drying and deprotection;

8. The method for synthesizing gonadorelin in full liquid phase according to claim 1, wherein the 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 a condensing agent, wherein the molar ratio of R4-Pyr-OH to the condensing agent to the organic base is 1: 1, and after the reaction is completed, filtering, washing and drying to obtain a compound 7;

9. The method for synthesizing gonadorelin in full liquid phase according to claim 1, wherein the 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-NH₂Carrying out a condensation reaction for the reaction unit, wherein R₁-Arg (pbf) -OH and H-Pro-Gly-NH₂In the molar ratio of 1: 1.05-2, adding organic base and condensing agent, 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;

10. The method for synthesizing gonadorelin in full liquid phase according to claim 1, wherein the 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;

step S11 specifically includes the following steps:

putting the compound 10 into a reactor, adding a lysate into the reactor, precipitating the compound by using frozen ether after the reaction is finished, filtering the solution, and collecting a solid to obtain a gonadorelin crude 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 gonadorelin in a full liquid phase.

Background

Gonadorelin, the chemical name of which is 5' -oxoprolinyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-glycyl-L-leucyl-L-arginyl-L-prolyl-glycinamide, the molecular formula of which is C₅₅H₇₅N₁₇O₁₃(ii) a The molecular weight is 1182.33; is a nonapeptide analogue of artificially synthesized gonadotropin releasing hormone (GnRH), is mainly used for clinically identifying and diagnosing male or female fertility disorder caused by hypothalamic or hypophyseal hypofunction, gonadal atrophic hypogonadism, galactorrhea amenorrhea, primary and secondary amenorrhea, menopause and premature menopause, pituitary tumor, organ injury of pituitary, actual hypothalamic dysfunction and the like.

In the prior art, the synthesis of gonadorelin is mainly a solid phase method, for example, chinese patent application 201710439767.8 reports a method for synthesizing gonadorelin by a solid phase synthesis method, the method uses rink amidea resin as a solid phase carrier, a condensing agent modulator is HBTU/HOBt/DIPEA, Fmoc amino acid as a monomer, piperidine as a deprotection reagent, and then coupling is performed one by one from a C end to an N end until a protected gonadorelin resin peptide chain is synthesized, and finally, gonadorelin is obtained by cracking. The method adopts piperidine which is an easily toxic reagent, the used Rink resin is expensive and cannot be recycled, and the last amino acid 5-oxyproline is not easily dissolved in an aprotic solvent which is commonly used for polypeptide synthesis. The Czech patent CZ2014976A3 adopts a similar amino acid monomer and Rink resin as a carrier synthesis method. US patent US4024248 mentions that gonadorelin analogues can be subjected to a fragment condensation method: the segments are condensed by an azide method, the condensed segments are not protected, side reactions are more, and the cost is higher. The invention CN202011087410.6 in China is a method for synthesizing gonadorelin by polypeptide solid-liquid combination, which is improved on the basis of a solid phase method, but still has the problem that impurities are difficult to purify. There is no report on the synthesis of gonadorelin by the all-liquid phase method.

Disclosure of Invention

Therefore, the invention aims to solve the technical problems of high cost, more solvents, easy-to-use toxic reagents, high environmental protection pressure and low purity of the crude product of the current mainstream solid-phase reaction, and provides a method for synthesizing gonadorelin in a full liquid phase. The purity of the crude product can reach more than 95 percent, thereby being beneficial to large-scale production.

The invention provides a method for synthesizing gonadorelin 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-Gly-Leu-OR₂；

S3, liquid phase synthesis of compound 3: H-Gly-Leu-OR₂；

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

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

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

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

S8, liquid phase synthesis of compound 8:R₄-Pyr-His(R₃)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-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)-Gly-Leu-Arg(pbf)-Pro-Gly-NH₂；

s11, preparing a gonadorelin crude product;

Preferably, step S1 specifically includes the following steps:

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-Gly-OH、H-Leu-OR₂Carrying out a condensation reaction for the reaction unit, R₁-Gly-OH and H-Leu-OR₂The molar ratio of the active agent to the condensing agent to the organic base is 1: 1.05-2, the active agent, the organic base and the condensing agent are added, the ratio of the H-Leu-OR2 to 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 solid is collectedObtaining 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:

the deprotection reagent comprises any one of trifluoroacetic acid, hydrogen chloride solution, 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:

Preferably, step S5 specifically includes the following steps:

Preferably, step S6 specifically includes the following steps:

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 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;

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;

step S9 specifically includes the following steps:

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:

Step S11 specifically includes the following steps:

The reagents used in the technical scheme are all common commercially available reagents; in the above technical solution, ether reagent is usually used for the operation of separating out or separating out solid, and includes 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 reagent which is extremely toxic and easy to produce toxic, the yield of the produced product is more than 82 percent, the purity of the gonadorelin crude product can reach more than 95 percent, the cost is greatly reduced, and the method 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 Compound 1 prepared in example 1 of the present invention;

FIG. 2 is an HPLC chromatogram of Compound 2 prepared in example 1 of the present invention;

FIG. 3 is an HPLC chromatogram of Compound 3 prepared in example 1 of the present invention;

FIG. 4 is an HPLC chromatogram of Compound 4 prepared in example 1 of the present invention;

FIG. 5 is an HPLC chromatogram of Compound 5 prepared in example 1 of the present invention;

FIG. 6 is an HPLC chromatogram of Compound 6 prepared in example 1 of the present invention;

FIG. 7 is an HPLC chromatogram of Compound 7 prepared in example 1 of the present invention;

FIG. 8 is an HPLC chromatogram of Compound 8 prepared in example 1 of the present invention;

FIG. 9 is an HPLC chromatogram of Compound 9 prepared in example 1 of the present invention;

FIG. 10 is an HPLC chromatogram of Compound 10 prepared in example 1 of the present invention;

FIG. 11 is an HPLC chromatogram of a crude alanrelin prepared in 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

Example 1

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

1.1 feeding:

the batch materials were dosed according to the material amounts of table 2.

TABLE 2

Material(s)	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 added, TEA was added to start the reaction, and the reaction was completed 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 ℃. The solid was collected, filled into a container and weighed. The HPLC detection conditions for compound 1 are:

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,

gradient: 0-30min, 70% B-90% B.

The results are shown in FIG. 1, with yields: 95.8%, purity: 87.6 percent.

2. Liquid phase synthesis of compound 2: Fmoc-Gly-Leu-OMe.HCl

2.1 charging

The materials were dosed as per table 3.

TABLE 3

Material(s)	Dosage of
		Fmoc-Gly-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-Gly-OH and HOSU in a reaction bottle, completely dissolving with DMF, 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 cold bathing for 5min, adding DCC, and detecting by HPLC to complete the reaction. After complete reaction, the reaction solution is filtered, 0.5M hydrochloric acid aqueous solution is used for precipitation, the filtered solid is washed to be neutral by purified water, the solid is dried at the temperature of 30 ℃, and the solid is collected, filled into a triangular flask and weighed.

The HPLC detection conditions for compound 2 were:

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,

gradient: 0-30min, 50% B-90% B.

The results are shown in FIG. 2, yield: 107.2%, purity: 95.7 percent.

3. Liquid phase synthesis of compound 3: H-Gly-Leu-OMe

3.1 feeding:

the materials were dosed as per table 4.

TABLE 4

Material(s)	Dosage of
		Fmoc-Gly-Leu-OMe.HCl	150mmol
Diethylamine	400ml
		Petroleum ether	1L

3.2 procedure

Accurately weighing Fmoc-Gly-Leu-OMe.HCl in a reaction bottle, adding diethylamine for reaction, and detecting complete reaction by HPLC. Concentrating to small amount, adding petroleum ether to precipitate solid, filtering, and vacuum drying. The HPLC detection conditions for compound 3 were:

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,

gradient: 0-30min, 3% B-90% B.

The results are shown in FIG. 3, with yields: 91.8%, purity: 94.6 percent.

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

4.1 feeding:

the materials were dosed as per table 5.

TABLE 5

Material(s)	Dosage of
		Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH	95.8mmol
BOP	105.4mmol
		TEA	105.4mmol
H-Gly-Leu-OMe	105.4mmol
		DMF	200ml
DCM	200ml
		0.5M hydrochloric acid solution	1L

4.2 procedure

Accurately weighing Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH and BOP in a reaction bottle, completely dissolving with DMF, cooling for 10min, adding DIEA, dissolving H-Gly-Leu-OMe with DCM, adding into the reaction for starting the reaction, detecting the reaction by HPLC, concentrating, precipitating with 0.5M hydrochloric acid, filtering, collecting the solid, washing with purified water to neutrality (detecting with pH test paper), and weighing. The HPLC detection conditions for compound 4 were:

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,

gradient: 0-30min, 80% B-90% B.

The results are shown in FIG. 4, yield: 92.3%, purity: 90 percent of

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

5.1 charging

The materials were dosed as per table 6.

TABLE 6

Material(s)	Dosage of
		Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe	88.4mmol
Diethylamine	500ml
		Petroleum ether	1L

5.2 procedure

Accurately weighing Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-OMe in a reaction bottle, adding diethylamine for reaction, detecting the reaction by HPLC, concentrating to a small amount, adding petroleum ether to precipitate a solid, filtering, and drying in vacuum.

The HPLC detection conditions for compound 5 were:

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,

gradient: 0-30min, 50% B-90% B.

The results are shown in FIG. 5, yield: 96.2%, purity: 88.6 percent.

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

6.1 charging

The materials were dosed as per table 7.

TABLE 7

Material(s)	Dosage of
		Fmoc-His(Trt)-OH	93.6mmol
HOBt	93.6mmol
		DCC	93.6mmol
H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe	85.1mmol
		Diethylamine	500ml
DMF	400ml
		0.5M hydrochloric acid solution	1L

6.2 procedure

Accurately weighing Fmoc-His (Trt) -OH, HOBt, H-Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-OMe in a reaction bottle, completely dissolving with DMF, cooling for 10min, adding DCC for reaction, filtering the reaction solution, pouring into the reaction bottle, adding diethylamine for reaction 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) -Leu-OMe into a reaction bottle, adding diethylamine for reaction for 20min, detecting by HPLC to complete the reaction, concentrating to a small amount, adding petroleum ether to precipitate a solid, filtering and drying.

The HPLC detection conditions for compound 6 were:

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,

gradient: 0-30min, 70% B-90% B.

The results are shown in FIG. 6, yield: 82.1%, purity: 92.8 percent.

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

7.1 charging

The materials were dosed as per table 8.

TABLE 8

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

7.2 procedure

Accurately weighing Boc-Pyr-OH and BOP in a reaction bottle, completely dissolving with DMF, performing cold bath for 10min, adding DIEA, dissolving H-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-OMe with DMF, adding into the reaction, starting the reaction, and detecting the reaction completely by HPLC; the reaction solution was filtered, the residue was washed twice with DMF, precipitated with 0.5M hydrochloric acid, filtered and the solid collected, then washed with purified water to neutral (pH paper test), dried and weighed.

The HPLC detection conditions for compound 7 were:

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,

gradient: 0-30min, 70% B-90% B.

The results are shown in FIG. 7, with yields: 85%, purity: 80.1 percent.

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

8.1 charging

Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe：59.4mmol

Methanol: 1110ml

2M NaOH：110ml

0.1M hydrochloric acid solution: 2L of

8.2 procedure

Weighing Boc-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-OMe, placing the mixture into methanol, stirring for 5min, completely dissolving, slowly adding 2M NaOH for reaction, starting the reaction, detecting the reaction by HPLC, adding a hydrochloric acid solution, precipitating, filtering, collecting a solid, washing the solid with purified water to be neutral (detecting by a pH test paper), drying and weighing. The HPLC assay conditions for compound 8 were:

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,

gradient: 0-30min, 70% B-90% B.

The results are shown in FIG. 8, yield: 83.9%, purity: 94.07 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

Material(s)	Dosage of
		H-Pro-Gly-NH2	150mmol
BOP	157.5mmol
		DIEA	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. Detecting reaction by HPLC, precipitating with 0.5M hydrochloric acid, filtering, collecting solid, washing with purified water to neutral (pH test paper), drying, adding diethylamine for reaction, detecting reaction by HPLC, concentrating to small amount, adding petroleum ether to precipitate solid, filtering, and vacuum drying. The HPLC detection conditions are 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,

gradient: 0-30min, 5% B-30% B.

Referring to fig. 9, the yield is 93%, and the purity: 95.5 percent.

10. Liquid phase synthesis of compound 10:

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

10.1 charging

The materials were dosed as in table 10.

Watch 10

Material(s)	Dosage of
		Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OH	49.8mmol
BOP	54.8mmol
		TEA	54.8mmol
H-Arg(pbf)-Pro-Gly-NH₂	54.8mmol
		DMF	200ml
0.5M hydrochloric acid solution	500ml

10.2 procedure

Accurately weighing Boc-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-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. HPLC detects the reaction is complete, 0.5M hydrochloric acid is added to precipitate a solid, the solid is collected by filtration, washed to neutrality by purified water (detected by pH test paper), dried and weighed. The HPLC detection conditions are 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,

gradient: 0-30min, 70% B-90% B.

Referring to fig. 10, the yield is 98.1%, and the purity: 94.8 percent

11. Synthesis of gonadorelin crude product

11.1 charging

Compound 10: 48.6mmol

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

11.2 procedure

Adding the compound 10 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. The HPLC detection conditions are 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,

gradient: 0-30min, 25% B-25% B.

The results are shown in FIG. 11, yield: 82.4% and 95.2% purity.

Example 2

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-Gly-Leu-OEt & HCl

2.1 charging

The materials were dosed as in table 11.

TABLE 11

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

The procedure was as in example 1. Yield: 106.2%, purity: 95.2 percent.

3. Liquid phase synthesis of compound 3: H-Gly-Leu-OEt

3.1 feeding:

the materials were dosed as in table 12.

TABLE 12

Material(s)	Dosage of
		Boc-Gly-Leu-OEt.HCl	150mmol
50％TFA/DCM	400ml
		Petroleum ether	1L

The procedure was as in example 1. Yield: 91.2%, purity: 90.5 percent.

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

4.1 feeding:

the materials were dosed as in table 13.

Watch 13

Material(s)	Dosage of
		Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH	95.8mmol
BOP	191.6mmol
		TEA	191.6mmol
H-Gly-Leu-OEt	191.6mmol
		DMF	200ml
DCM	200ml
		0.5M hydrochloric acid solution	1L

The procedure was as in example 1. Yield: 91.8%, purity: 80 percent of

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

5.1 charging

The material charges according to table 14.

TABLE 14

Material(s)	Dosage of
		Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt	88.4mmol
Diethylamine	500ml
		Petroleum ether	1L

The procedure was as in example 1. Yield: 95.8%, purity: 84.8 percent.

6. Liquid phase synthesis of compound 6: H-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-OEt6.1 feeding

The materials were dosed as per table 15.

Watch 15

Material(s)	Dosage of
		Fmoc-His(Boc)-OH	170.2mmol
HOBt	170.2mmol
		DCC	170.2mmol
H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt	170.2mmol
		Diethylamine	500ml
DMF	400ml
		0.5M hydrochloric acid solution	1L

The procedure was as in example 1. Yield: 82.0%, purity: 81.5 percent.

7. Liquid phase synthesis of compound 7: Fmoc-Pyr-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-OEt

7.1 charging

The charges were carried out with the materials according to Table 16.

TABLE 16

Material(s)	Dosage of
		Fmoc-Pyr-OH	139.8mmol
BOP	139.8mmol
		Diethylamine	139.8mmol
H-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt	69.9mmol
		DMF	350ml
0.5M hydrochloric acid solution	1L

The procedure was as in example 1. Yield: 85%, purity: 85.3 percent.

8. Liquid phase synthesis of compound 8: Fmoc-Pyr-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-OH

8.1 charging

Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt：59.4mmol

Methanol: 1110ml

2M NaOH：1110ml

0.1M hydrochloric acid solution: 2L of

The procedure was as in example 1. Yield: 83.5%, purity: 88.6 percent.

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

9.1 charging

The materials were dosed as in table 17.

TABLE 17

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

The procedure was as in example 1. Yield 93%, purity: 82 percent.

10. Liquid phase synthesis of compound 10:

Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-Arg-Pro-Gly-NH₂

10.1 charging

The charges were carried out according to the materials in Table 18.

Watch 18

Material(s)	Dosage of
		Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OH	49.8mmol
BOP	99.6mmol
		TEA	99.6mmol
H-Arg-Pro-Gly-NH₂	99.6mmol
		DMF	200ml
0.5M hydrochloric acid solution	500ml

The procedure was as in example 1. Yield 97.8%, purity: 81.1 percent

11. Synthesis of gonadorelin crude product

11.1 charging

Compound 10: 48.7mmol

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

The procedure was as in example 1. Yield: 82.1 percent and the purity is 95.1 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.

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