CN112175042B

CN112175042B - Method for synthesizing Etelcalcetide

Info

Publication number: CN112175042B
Application number: CN201910595277.6A
Authority: CN
Inventors: 陶志强; 姚志军; 宓鹏程; 陶安进; 袁建成
Original assignee: Hybio Pharmaceutical Co Ltd
Current assignee: Hybio Pharmaceutical Co Ltd
Priority date: 2019-07-03
Filing date: 2019-07-03
Publication date: 2022-05-31
Anticipated expiration: 2039-07-03
Also published as: CN112175042A; WO2021000543A1

Abstract

The invention relates to the field of polypeptide synthesis, in particular to a synthesis method of Etelcalcetide. The invention adopts beta-S (Trt) -D-Arg (Boc)3-OH as a raw material, cysteine to replace alanine in a peptide sequence, and adopts an NCL (natural chemical ligation) method to carry out coupling reaction, the invention improves the defects of the existing liquid phase method, and adopts the intramolecular migration reaction from S to N to complete coupling according to the unique characteristics of the peptide sequence, thereby improving the coupling efficiency between peptide fragments, and the total yield of the method is 71.3%. Therefore, the invention overcomes the defect of difficult coupling among fragments under the action of the traditional coupling reagent, reduces the production cost and is suitable for large-scale production.

Description

Method for synthesizing Etelcalcetide

Technical Field

The invention relates to the field of polypeptide synthesis, in particular to a synthesis method of Etelcalcetide.

Background

Etelcalcetide is a novel calcium mimetic developed by AMGEN INC, and is mainly used as a polypeptide drug for secondary hyperparathyroidism in hemodialysis treatment of adult patients with chronic kidney diseases. Marketed in 2017 in the united states on day 07 of month 2 under the trade name Parsabiv. The main chain of Etelcalcetide consists of seven D-amino acids, with the side chains linked to the L-cysteine by a disulfide bond. The peptide sequence is shown as formula I:

solid-phase coupling adopted by Etelcalcet requires the use of resin, and the large-scale production is limited. In addition, the amino acid is usually required to be excessive (3-5 equivalents) during coupling, and the Etelcalcetide mainly takes expensive D-type amino acid, so that the solid-phase synthesis method is high in cost and limited in large-scale production.

The coupling between the amino acids of patent CN106928321 and WO2016154580A1 is activated by using conventional coupling reagents (PyBop, HATU, DIC or EDC, etc.), but the coupling efficiency of the method for the fragments is usually low, resulting in low yield.

Disclosure of Invention

In view of the above, the invention provides a synthesis method of Etelcalcetide. The method adopts the intramolecular migration reaction from S to N to complete the coupling, thereby improving the coupling efficiency between peptide fragments, and the total yield is 71.3%. Overcomes the defect of difficult coupling among fragments under the action of the traditional coupling reagent, reduces the production cost and is suitable for large-scale production.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a synthesis method of Etelcalcetide, which takes beta-S (Trt) -D-Arg (Boc)3-OH as a raw material, substitutes cysteine for alanine in a peptide sequence, and adopts an NCL method to carry out coupling reaction to prepare the Etelcalcetide.

The method specifically comprises the following steps:

step 1: with beta-S (Trt) -D-Arg (Boc)₃OH and thiophenol (PhSH) are used as raw materials, and a compound 1 is obtained through coupling reaction in the presence of a solvent;

and 2, step: Ac-D-Cys (Acm) -OH and HONb are used as raw materials, activated by a coupling agent in the presence of a solvent, and subjected to dehydration condensation with H-Ala-OH to obtain a compound 2;

and step 3: taking a compound 2 and thiophenol (PhSH) as raw materials, and carrying out condensation reaction in the presence of a solvent and a coupling agent to obtain a compound 3;

and 4, step 4: Boc-D-Cys (Trt) -OH, H-D-Arg (Pbf) -NH₂HCI is taken as a raw material, and a dipeptide fragment, namely a compound 4, is obtained through condensation reaction in the presence of a solvent and a coupling agent;

and 5: performing deprotection reaction on the compound 4 in the presence of a lysate to obtain a compound 5;

step 6: cracking a compound 5 and a compound 1 serving as raw materials to obtain a compound 6 by NCL reaction in the presence of a solvent, a buffer solution, TCEP and trifluoroethanol;

and 7: cracking a compound 6 and a compound 1 serving as raw materials to obtain a compound 7 through NCL reaction in the presence of a solvent, a buffer solution, TCEP and trifluoroethanol;

and 8: cracking a compound 7 and a compound 1 serving as raw materials to obtain a compound 8 through NCL reaction in the presence of a solvent, a buffer solution, TCEP and trifluoroethanol;

and step 9: taking a compound 8 and a compound 3 as raw materials, carrying out NCL reaction in the presence of a solvent, a buffer solution, TCEP and trifluoroethanol, and then carrying out desulfurization reaction and purification in the presence of an initiator and tBuSH to obtain a compound 9;

step 10: the compound 9 and L-Cys-OH are used as raw materials, and Etecalacetide is obtained by iodine oxidation and purification.

In some embodiments of the present invention, the solvent in step 1 comprises one or a mixture of two of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, and dichloromethane; preferably dichloromethane;

step 1 beta-S (Trt) -D-Arg (Boc)₃The molar ratio of-OH to thiophenol (PhSH) is 1: 1.0-1.5;

the temperature of the coupling reaction in the step 1 is 20-30 ℃; the coupling reaction time is 2-10 h; preferably 5 h; the coupling agent adopted in the coupling reaction comprises a composition of DIC and a compound A, and a composition of DIPEA, the compound A and a compound B, wherein the compound A is HOAt or HOBt, and the compound B is one or a composition of more than two of PyAOP, PyBOP, HATU, HBTU, TBTU or EDC; preferably EDC/HOBT/DIPEA; further, the molar ratio of the coupling reagent components was EDC/HOBT/DIPEA ═ 1:1: 2.

In some embodiments of the invention, the molar ratio of Ac-D-Cys (Acm) -OH to HONb in step 2 is 1:1.0 to 1.5;

the solvent selected for activation in the step 2 comprises one or a composition of more than two of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform and dichloromethane; preferably dichloromethane;

the activation temperature in the step 2 is 20-30 ℃, and the activation time is 1-5 h; preferably 3 h;

the coupling agent adopted for activation in the step 2 is DIC or a composition of DIPEA and a compound B, and the compound B is one or a composition of more than two of PyAOP, PyBOP, HATU, HBTU, TBTU or EDC; preferably DIC;

the solvent selected for dehydration condensation in the step 2 comprises one or a mixture of more than two of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane and water; preferably a mixed solvent of water and acetonitrile; further, the ratio of water to acetonitrile is 2: 1;

in the step 2, the temperature of the dehydration condensation is 20-30 ℃, and the time of the dehydration condensation is 1-3 h; preferably for 2 h;

the Acm protecting group in Ac-D-Cys (Acm) -OH may also be replaced with a Trt protecting group.

In some embodiments of the invention, the molar ratio of compound 2 to thiophenol (PhSH) in step 3 is 1:1.0 to 1.5;

in step 3, the solvent adopted by the condensation reaction comprises one or a composition of more than two of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform and dichloromethane; preferably dichloromethane;

in the step 3, the temperature of the condensation reaction is 20-30 ℃, and the time of the condensation reaction is 2-10 h; preferably 5 h;

in the step 3, the coupling agent is a composition of DIC and a compound A, or a composition of DIPEA, the compound A and a compound B, wherein the compound A is HOAt or HOBt, and the compound B is PyAOP, PyBOP, HATU, HBTU, TBTU or EDC; preferably an EDC/HOBT/DIPEA composition; further, the molar ratio of EDC/HOBT/DIPEA is 1:1: 2.

In some embodiments of the invention, Boc-D-Cys (Trt) -OH, and H-D-Arg (Pbf) -NH in step 4₂The molar ratio of HCI is 1: 1.0-1.5;

the temperature of the condensation reaction in the step 4 is 20-30 ℃, and the time of the condensation reaction is 2-10 h; preferably 5 h;

the solvent in the step 4 comprises one or a mixture of more than two of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform and dichloromethane; preferably dichloromethane;

in the step 4, the coupling agent is a composition of DIC and a compound A, and a composition of DIPEA, the compound A and a compound B, wherein the compound A is HOAt or HOBt, and the compound B is one or a mixture of more than two of PyAOP, PyBOP, HATU, HBTU, TBTU or EDC; preferably EDC/HOBT/DIPEA; further, the molar ratio of EDC/HOBT/DIPEA is 1:1: 2;

in some embodiments of the invention, the lysis solution in step 5 is TFA, H₂A combination of O and TIS; TFA, H₂The volume ratio of O to TIS is 95:2.5: 2.5;

the temperature of the deprotection reaction in the step 5 is 20-30 ℃, and the time of the deprotection reaction is 2-4 h; preferably 3 h;

in some embodiments of the invention, in steps 6 to 9, the molar ratio of each of the compound 5, the compound 6, the compound 7 and the compound 8 to the compound 1 is 1.0 to 1.1: 1;

in the step 6-9, the buffer solution is 6.0M guanidine hydrochloride (Gn. HCl) and 0.2M Na₂HPO₄The pH value of the mixed solution is 5.6-7.6; preferably the pH is 6.6;

in the step 6-9, the solvent comprises one or a mixture of more than two of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform and dichloromethane; preferably acetonitrile;

in the step 6-9, the temperature of the NCL reaction is 20-30 ℃, and the time of the NCL reaction is 2-6 h; preferably 4 h;

in the step 6-9, the consumption of TCEP and trifluoroethanol is 5-15 equivalents of the raw materials; preferably 10 equivalents;

the cracking solution adopted in the cracking in the step 6-8 is TFA and H₂O, TIS composition, TFA: H₂The volume ratio of O to TIS is 95:2.5: 2.5;

the temperature of the pyrolysis in the step 6-8 is 20-30 ℃, and the time of the pyrolysis is 2-4 h; preferably 3 h.

In some embodiments of the invention, the initiator in step 9 is VA-044; the using amount of the initiator is 0.01-0.03 equivalent of that of the compound 3; preferably 0.02 equivalents; the dosage of tBuSH is 1-3 equivalents of the compound 3; preferably 2 equivalents;

in the step 9, the temperature of the desulfurization reaction is 20-30 ℃, and the time of the desulfurization reaction is 2-4 h; preferably for 3 hours;

the purification in step 9 is performed by reversed phase high pressure liquid chromatography comprising: using reverse octadecylsilane as stationary phase, using 0.1% trifluoroacetic acid water solution/acetonitrile as mobile phase, collecting maximum peak fraction, concentrating under reduced pressure at 30 deg.C to 1/4 volume, and lyophilizing.

In some embodiments of the present invention, the solvent used in the iodine oxidation in step 10 is one or a mixture of more than two of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane, methanol, ethanol and water; preferably a mixed solvent of methanol and water; further, the ratio of methanol to water is 1: 5;

in the step 10, adjusting the pH value of iodine oxidation to 2-4 by adopting glacial acetic acid; preferably the pH is 3;

in the step 10, the amount of the iodine is 1-3 equivalent of the raw material; preferably 2 equivalents;

in the step 10, the temperature of the iodine oxidation is 20-30 ℃, and the time of the iodine oxidation is 1-5 h; preferably for 3 hours;

in the step 10, the purification adopts reversed phase high pressure liquid chromatography; the reversed phase high pressure liquid chromatography comprises: using reverse octadecylsilane as stationary phase, using 0.1% trifluoroacetic acid water solution/acetonitrile as mobile phase, collecting maximum peak fraction, concentrating under reduced pressure at 30 deg.C to 1/4 volume, and lyophilizing.

Solid-phase coupling of Etelcalcetide requires the use of resin, and large-scale production is limited. In addition, the amino acid is usually required to be excessive (3-5 equivalents) during coupling, and the Etelcalcetide mainly takes expensive D-type amino acid, so that the solid-phase synthesis method is high in cost and limited in large-scale production. The traditional fragment method is used for the large steric hindrance between the fragments, which results in the coupling efficiency being generally low, resulting in the yield being too low, the yield of coupling between two fragments of the comparative example being only 43.2%, and the yield being not higher than 5% due to the loss of synthesis of the fragment itself.

The NCL (native chemical ligation) method is an efficient method for assembling two or more unprotected peptide segments to construct a protein. The invention adopts beta-S (Trt) -D-Arg (Boc)3-OH as a raw material, cysteine to replace alanine in a peptide sequence, and adopts an NCL (natural chemical ligation) method to carry out coupling reaction, the invention improves the defects of the existing liquid phase method, and adopts the intramolecular migration reaction from S to N to complete coupling according to the unique characteristics of the peptide sequence, thereby improving the coupling efficiency between peptide fragments, and the total yield of the method is 71.3%. Therefore, the invention overcomes the defect of difficult coupling among fragments under the action of the traditional coupling reagent, reduces the production cost and is 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.

FIG. 1 shows a flow chart of a preparation method provided by the present invention;

FIG. 2 shows an HPLC chromatogram of Compound 6;

FIG. 3 shows a mass spectrum of Compound 6;

FIG. 4 shows an HPLC chromatogram of Compound 8;

FIG. 5 shows the mass spectrum of Compound 8;

FIG. 6 shows a chromatogram of HPLC of Compound 9;

FIG. 7 shows a mass spectrum of Compound 9;

FIG. 8 shows a spectrum of an Etelcalcetide HPLC;

FIG. 9 shows a fine peptide mass spectrum of Etelcalcetide;

FIG. 10 shows the spectrum of a comparative example Etelcalcetide HPLC;

fig. 11 shows the mass spectrum of comparative example etelcalcide.

Detailed Description

The invention discloses a synthesis method of Etelcalcetide, and a person skilled in the art can appropriately improve process parameters by referring to the content of the Etelcalcetide. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the method and application of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the method and application described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

The NCL (native chemical ligation) method is an efficient method for assembling two or more unprotected peptide segments to construct a protein. Therefore, in order to reduce the cost and overcome the problem of difficult coupling between fragments, the invention adopts beta-S (Trt) -D-Arg (Boc) according to the characteristics of the peptide sequence of the Etelcalcetide₃-OH is used as raw material, cysteine is used for replacing alanine in a peptide sequence, and a coupling reaction is carried out by adopting an NCL (natural chemical ligation) method. As shown in particular in figure 1.

The invention uses beta-S (Trt) -D-Arg (Boc)₃ Synthesizing thioester compound 1 by using-OH and thiophenol as raw materials under the action of a coupling reagent. Ac-D-Cys (Acm) -OH and H-Ala-OH are used as raw materials to synthesize a segment compound 2 under the action of a coupling reagent, and then the segment compound and thiophenol are synthesized into a thioester compound 3 under the action of the coupling reagent. Boc-D-Cys (Trt) -OH, H-D-Arg (Pbf) -NH₂HCI is used as a raw material to synthesize a dipeptide fragment compound 4 under the action of a coupling reagent, and a compound 5 is obtained after deprotection. Compound 5 and thioester compound 1 were reacted in a buffer solution with NCL by the action of TCEP and trifluoroethanol to obtain compound 6. Sequential coupling of two thioester compounds 1 by means of the NCL method was continued to give compoundsAnd (3) substance 8. After NCL reaction of the compound 8 and the thioester compound 2, desulfurization reaction is carried out under the action of water-soluble azo initiators VA-04, TCEP and tert-butyl mercaptan to obtain a compound 9, and the compound 9 and L-Cys-OH are subjected to iodoxidation to obtain the Etecalacetide.

Step 1: synthesis of Compound 1 (. beta. -S (Trt) -D-Arg (Boc)₃-SPh)

And 2, step: synthesis of Compound 2 (Ac-D-Cys (Acm) -D-Ala-OH)

And step 3: synthesis of Compound 3 (Ac-D-Cys (Acm) -D-Ala-SPh)

And 4, step 4: synthesis of Compound 4 (Boc-D-Cys (Trt) -D-Arg (Pbf) -NH)₂)

And 5: synthesis of Compound 5 (H-D-Cys-D-Arg-NH)₂)

Step 6: synthesis of Compound 6 (H-D-Arg (. beta. -SH) -D-Cys-D-Arg-NH)₂)

And 7: synthesis of Compound 7 (H-D-Arg (. beta. -SH) -D-Cys-D-Arg-NH)₂)

And step 8: synthesis of Compound 8 (H-D-Arg (. beta. -SH) - - -D-Arg (. beta. -SH) -D-Arg (beta-SH) -D-Cys-D-Arg-NH)₂)

And step 9: synthesis of Compound 9 (Ac-D-Cys (Acm) -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH)₂)

Step 10: synthesis of Etecalacetide

Step 1 mainly uses beta-S (Trt) -D-Arg (Boc)₃the-OH and thiophenol (PhSH) are used as raw materials, and the molar ratio is 1: 1.0-1.5. The coupling reaction was carried out at room temperature (25 ℃ C. + -5 ℃ C.) to obtain Compound 1. The solvent used includes tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane and the like, preferably dichloromethane. The coupling reaction time is 2-10 hours, preferably 5 hours, and the coupling agent is a composition of DIC and a compound A or a composition of DIPEA, a compound A and a compound B, wherein the compound A is HOAt or HOBt, and the compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, EDC. Preferably an EDC/HOBT/DIPEA composition. Further, the coupling reagent component ratio was EDC/HOBT/DIPEA ═ 1:1: 2.

In the step 2, Ac-D-Cys (Acm) -OH and HONb are used as raw materials, the molar ratio is 1: 1.0-1.5, the raw materials are activated by a coupling agent at room temperature (25 +/-5 ℃), and the activated raw materials and H-Ala-OH are subjected to dehydration condensation under the action of DIPEA to obtain a compound 2. The solvent used for the activation reaction includes tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane and the like, and dichloromethane is preferred. The activation reaction is carried out at room temperature (25 ℃ C. + -5 ℃ C.) for 1 to 5 hours, preferably 3 hours. The activating coupling agent is DIC or a combination of DIPEA and a compound B, the compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, EDC is preferable DIC. The solvent for dehydration condensation reaction with H-Ala-OH includes tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane, water, etc., preferably a mixed solvent of water and acetonitrile, and further the ratio of water to acetonitrile is 2: 1. The coupling reaction is carried out at room temperature (25 ℃ C.. + -. 5 ℃ C.) for 1 to 3 hours, preferably 2 hours. The Acm protecting group in Ac-D-Cys (Acm) -OH may also be replaced with a Trt protecting group.

In the step 3, a compound 2 and thiophenol (PhSH) are used as raw materials, the molar ratio is 1: 1.0-1.5, and the condensation reaction is carried out on a coupling agent at room temperature (25 +/-5 ℃). The solvent selected includes tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane, etc., preferably dichloromethane. The reaction time is 2-10 hours, preferably 5 hours, and the coupling agent is DIC and compound A composition or DIPEA and compound A and compound B composition, wherein compound A is HOAt or HOBt, and compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, EDC. Preferably EDC/HOBT/DIPEA compositions. Further, the molar ratio of the coupling reagent components was EDC/HOBT/DIPEA ═ 1:1: 2.

In step 4, Boc-D-Cys (Trt) -OH, H-D-Arg (Pbf) -NH₂HCI is used as a raw material, the molar ratio is 1: 1.0-1.5, and the dipeptide fragment compound 4 is obtained by condensation reaction through a coupling reagent at room temperature (25 +/-5 ℃). The solvent includesSolvents such as tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform and dichloromethane, preferably dichloromethane. The reaction time is 2-10 hours, preferably 5 hours, and the coupling agent is DIC and compound A composition or DIPEA and compound A and compound B composition, wherein compound A is HOAt or HOBt, and compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, EDC. Preferably EDC/HOBT/DIPEA compositions. Further, the molar ratio of the coupling reagent components was EDC/HOBT/DIPEA ═ 1:1: 2.

And (5) performing deprotection reaction on the compound in the step 5 through a lysate. With lysis buffer TFA: H₂The lysis was carried out with a volume ratio of O to TIS of 95:2.5: 2.5. The room temperature (25 ℃ C. + -. 5 ℃ C.) cracking time is 2-4 hours, preferably 3 hours.

In the step 6, the compound 5 and the compound 1 are in a buffer solution with the molar ratio of 1.0-1.1: 1, and are subjected to NCL reaction under the action of TCEP and trifluoroethanol and then are cracked to obtain the compound 6. The buffer solution is 6.0M guanidine hydrochloride (Gn. HCl),0.2MNa2HPO4 mixed solution, and its pH value is 5.6-7.6, and the preferable pH value is 6.6. The cosolvent solvent selected in the reaction is tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane, etc., preferably acetonitrile. The reaction time is 2 to 6 hours, preferably 4 hours, at room temperature (25 ℃ C. + -5 ℃ C.). The catalyst TCEP/trifluoroethanol is used in an amount of 5 to 15 equivalents, preferably 10 equivalents, based on the substrate. And (3) carrying out deprotection reaction on the compound 6 through a cracking solution. With lysis solution TFA: H₂And (3) performing cracking with the volume ratio of the O to the TIS of 95:2.5: 2.5. The room temperature (25 ℃ C. + -. 5 ℃ C.) cracking time is 2-4 hours, preferably 3 hours.

Step 7 describes the same procedure as step 6, followed by coupling of compound 1 to obtain compound 7.

Step 8 describes the

same steps

6 and 7, and compound 8 is obtained by coupling compound 1 sequentially.

The NCL reaction conditions in step 9 are as described for step 6. After the NCL reaction is finished, VA-044 is used as an initiator for desulfurization reaction. Finally preparative purification by reverse phase HPLC gave compound 9. The amount of the initiator used is 0.01 to 0.03 equivalent, preferably 0.02 equivalent, to the compound 3. The proton is provided as tBuSH in an amount of 1 to 3 equivalents, preferably 2 equivalents, to compound 3. The desulfurization reaction is carried out at room temperature, and the desulfurization reaction time is 2-4 hours, preferably 3 hours. The purification step can be performed by reverse phase high pressure liquid chromatography. Further, the reverse phase high pressure liquid chromatography comprises: using reverse octadecylsilane as stationary phase and 0.1% trifluoroacetic acid aqueous solution/acetonitrile as mobile phase, collecting maximum peak fraction, concentrating under reduced pressure at 30 deg.C to 1/4 volume, and lyophilizing.

In the step 10, the compound 9 and L-Cys-OH are oxidized by iodine to obtain the Etecalacetide. The solvent selected for the oxidation with iodine is tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, methylene chloride, methanol, ethanol, water, etc., preferably a mixed solvent of methanol and water, and further the ratio of methanol to water is 1: 5. Adjusting the pH value to be within a range of 2-4 by adopting glacial acetic acid, and preferably adjusting the pH value to be 3. The amount of iodine is 1-3 equivalents, preferably 2 equivalents, of the substrate. And (3) oxidizing at room temperature for 1-5 hours, preferably 3 hours. The purification step can be performed by reversed phase high pressure liquid chromatography. Further, the reversed-phase high-pressure liquid chromatography comprises: using reverse octadecylsilane as stationary phase, using 0.1% trifluoroacetic acid water solution/acetonitrile as mobile phase, collecting maximum peak fraction, concentrating under reduced pressure at 30 deg.C to 1/4 volume, and lyophilizing.

The raw materials and reagents used in the synthesis method of the Etelcalcetide provided by the invention are all commercially available.

The invention is further illustrated by the following examples:

example 1: synthesis of Compound 1 (. beta. -S (Trt) -D-Arg (Boc)₃-SPh)

Reacting beta-S (Trt) -D-Arg (Boc)₃-OH (30.0g, 40mmol), PhSH (5.3g, 48mmol), EDC (13.8g, 72mmol) and HOBt (9.43g, 72mmol) were added to 300mL of dichloromethane and the solution stirred, cooled in an ice bath to below 5 ℃ and DIPEA (37.7mL, 216mmol) was added dropwise, the temperature being controlled not to exceed 10 ℃. After the addition was complete, stirring was continued for 5 hours at room temperature and the reaction was monitored by TLC. After completion of the reaction, purified water (300mL) was added to the reaction mixture, and after stirring for 15 minutes, the mixture was allowed to stand for liquid separation, and the lower organic phase was collected. The organic phase was concentrated under reduced pressure at 30 ℃ and the residue was dissolved with ethyl acetate (300mL) under stirring, washed successively with purified water (300mL), saturated sodium bicarbonate solution (300mL) and saturated sodium chloride solution (300mL), the organic phase was collected and dried over anhydrous sodium sulfate overnight. After filtration, the filtrate was concentrated to 60mL, and n-hexane (240mL) was added slowly dropwise. White solid is separated out, and after stirring is continued for 1 hour, the mixture is kept stand still at the temperature of 0 ℃ for crystallization overnight. After filtration, vacuum drying at 40 ℃ gave 30.6g of a white solid in 91.2% yield.

Example 2: synthesis of Compound 2 (Ac-D-Cys (Acm) -D-Ala-OH)

Ac-D-Cys (Acm) -OH (3.5g, 15mmol), HONb (3.0g, 16.5mmol) were dissolved in 35mL DCM and the solution stirred, cooled to below 5 ℃ in an ice bath, DIC (2.8mL, 18mmol) was slowly added, stirring was continued at room temperature for 3 hours, and the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was filtered, concentrated under reduced pressure, and the residue was dissolved in 35mL of acetonitrile, 17.5mL of purified water was added, then H-Ala-OH (1.6g, 18mmol) was added, and stirring was continued, and DIPEA (4.0mL, 22.5mmol) was added dropwise. Stir at room temperature for 2 hours and TCL monitors the reaction. After the reaction, the mixture was filtered and concentrated. The residue was dissolved in 35mL of ethyl acetate with stirring, and then a saturated citric acid solution was added thereto to adjust the pH to 3 to 4, followed by standing to separate the mixture. The organic phase was washed with a saturated sodium bicarbonate solution (35mL) and a saturated aqueous salt solution (35mL) in this order, allowed to stand for separation, and dried over anhydrous sodium sulfate overnight. And (5) filtering. Concentrated to 10mL under reduced pressure at 40 ℃. N-hexane (40mL) was added dropwise, cooled to 0 ℃ and crystallized overnight. Filtration and vacuum drying gave 4.35g of a white solid in 95.0% yield.

Example 3: synthesis of Compound 3 (Ac-D-Cys (Acm) -D-Ala-SPh)

Ac-D-Cys (Acm) -D-Ala-OH (4.35g, 14.2mmol), H-D-Arg (Pbf) -NH 2. HCI (1.87g, 18mmol), EDC (4.2g, 21.3mmol) and HOBt (2.88g, 21.3mmol) obtained in example 2 were added to 50mL of dichloromethane and stirred to dissolve the solution, the ice bath was cooled to below 5 ℃, DIPEA (10.9mL, 63.9mmol) was added dropwise and the temperature was controlled not to exceed 10 ℃. After the addition was complete, stirring was continued for 5 hours at room temperature and the reaction was monitored by TLC. After completion of the reaction, purified water (50mL) was added to the reaction mixture, and after stirring for 15 minutes, the mixture was allowed to stand for liquid separation, and the lower organic phase was collected. The organic phase was concentrated under reduced pressure at 40 ℃ and the residue was dissolved with ethyl acetate (50mL) under stirring, washed successively with purified water (50mL), saturated sodium bicarbonate solution (50mL), and saturated sodium chloride solution (50mL), the organic phase was collected and dried over anhydrous sodium sulfate overnight. Filtration was carried out, the filtrate was concentrated to 15mL, and n-hexane (45mL) was slowly added dropwise. White solid is separated out, and after stirring is continued for 1 hour, the mixture is kept stand still for crystallization at the temperature of 0 ℃ overnight. After filtration, vacuum drying at 40 ℃ gave 5.51g of a white solid with a yield of 97.7%.

Example 4: synthesis of Compound 4 (Boc-D-Cys (Trt) -D-Arg (Pbf) -NH)₂)

Boc-D-Cys (Trt) -OH (4.64g, 10mmol), H-D-Arg (Pbf) -NH₂HCI (5.55g, 12mmol), EDC (2.95g, 15mmol) and HOBt (1.96g, 15mmol) were added to 30mL of dichloromethane and the solution stirred, cooled to below 5 ℃ in an ice bath, and DIPEA (7.7mL, 45mmol) was added dropwise, the temperature being controlled not to exceed 10 ℃. After the addition was complete, stirring was continued for 5 hours at room temperature and the reaction was monitored by TLC. After completion of the reaction, purified water (30mL) was added to the reaction mixture, and after stirring for 15 minutes, the mixture was allowed to stand for liquid separation, and the lower organic phase was collected. The organic phase was concentrated under reduced pressure at 30 ℃ and the residue was dissolved with ethyl acetate (30mL) under stirring, washed with purified water (30mL), saturated sodium bicarbonate solution (30mL) and saturated sodium chloride solution (30mL) in this order, and the organic phase was collected and dried over anhydrous sodium sulfate overnight. Filtered and concentrated, and the residue was taken up in 45mL of methyl tert-ether and warmed to 50 ℃ with stirring to dissolve it clear. Naturally cooling to room temperature to precipitate a white solid, and continuously standing at 0 ℃ for crystallization overnight. After filtration, vacuum drying at 40 ℃ gave 8.50g of a white solid with a yield of 97.8%.

Example 5: synthesis of Compound 5 (H-D-Cys-D-Arg-NH)₂)

Example 4 toTo this compound 4 was added 30mL of prefreezed lysis buffer (TFA: H)₂TIS 95:2.5:2.5), and stirred at room temperature for 3 hours. The reaction solution was poured into 240mL of frozen ether for precipitation, centrifuged, and washed twice with ether. Nitrogen blow drying gave 2.70g of a white solid in 100% yield.

Example 6: synthesis of Compound 6 (H-D-Arg (. beta. -SH) -D-Cys-D-Arg-NH)₂)

The H-D-Cys-D-Arg-NH synthesized in example 5 was added₂(2.70g, 9.8mmol) and beta-S (Trt) -D-Arg (Boc) synthesized in example 1₃-SPh (7.82g, 9.3mmol) was added to 20mL acetonitrile and stirred, and 60mL buffer solution (6.0M Gn · HCl,0.2M Na2HPO4, pH 6.6) was added and the solution was stirred. TCEP (23.2g, 93mmol) and TFE (9.3g, 93mmol) were added, stirred at room temperature for 4 hours and the reaction monitored by TLC. After the reaction, the mixture was concentrated, and the turbid solution was dissolved in dichloromethane (50mL) with stirring. The layers were separated by standing, and the organic phase was washed three times with purified water (50mL) and saturated sodium chloride solution (50 mL. times.2) in this order. The organic phase was collected, concentrated and added to 30mL of prefreezed lysate (TFA: H)₂TIS 95:2.5:2.5), and stirred at room temperature for 3 hours. The reaction solution was poured into 240mL of frozen ether for precipitation, centrifuged, and washed twice with ether. Nitrogen blow drying to obtain 4.16g of white solid with purity of 96.9% (see figure 1), yield of 96.2%, MS [ M + H ]]465 (see fig. 2).

Example 7: synthesis of Compound 7 (H-D-Arg (. beta. -SH) -D-Cys-D-Arg-NH)₂)

Compound 6(4.16g, 9.0mmol) synthesized in example 6 and β -S (Trt) -D-Arg (Boc) synthesized in example 1 were added₃-SPh (7.20g, 8.6mmol) was added to 20mL acetonitrile, stirred, and 60mL buffer solution (6.0M Gn · HCl,0.2M Na2HPO4, pH 6.6) was added and the solution was stirred. TCEP (22.5g, 90mmol) and TFE (9.0g, 90mmol) were added, stirred at room temperature for 4 hours and the reaction monitored by TLC. After the reaction, the mixture was concentrated, and the turbid solution was dissolved in dichloromethane (50mL) with stirring. The layers were separated by standing, and the organic phase was washed three times with purified water (50mL) and saturated sodium chloride solution (50 mL. times.2) in this order. The organic phase was collected, concentrated and added to 30mL of prefreezed lysate (TFA: H)₂TIS 95:2.5:2.5), stirred at room temperature for 3 hours. The reaction solution was poured into 240mL of frozen ether for precipitation, centrifuged, and washed twice with ether. Nitrogen is present inAir-dried to obtain 5.40g of white solid with a yield of 95.8%.

Example 8:

synthesis of Compound 8

(H-D-Arg(β-SH)--D-Arg(β-SH)-D-Arg(β-SH)-D-Cys-D-Arg-NH₂)

Compound 7(5.40g, 8.2mmol) synthesized in example 7 and β -S (Trt) -D-Arg (Boc) synthesized in example 1 were added₃-SPh (6.56g, 7.8mmol) was added to 20mL acetonitrile, stirred, and 60mL buffer solution (6.0M Gn · HCl,0.2M Na2HPO4, pH 6.6) was added and the solution was stirred. TCEP (19.5g, 78mmol) and TFE (7.8g, 78mmol) were added, stirred at room temperature for 4 hours and the reaction monitored by TLC. After the reaction, the mixture was concentrated, and the turbid solution was dissolved in dichloromethane (50mL) with stirring. The layers were separated by standing, and the organic phase was washed three times with purified water (50mL) and saturated sodium chloride solution (50 mL. times.2) in this order. The organic phase was collected, concentrated and added to 30mL of prefreezed lysate (TFA: H)₂TIS 95:2.5:2.5), stirred at room temperature for 3 hours. The reaction solution was poured into 240mL of frozen ether for precipitation, centrifuged, and washed twice with ether. Blowing dry with nitrogen to give 6.16g of a white solid with a purity of 93.9% (see FIG. 3), yield 93.9%, MS [ M + H ]]841 (see FIG. 4).

Example 9:

synthesis of Compound 9

(Ac-D-Cys(Acm)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂)

Compound 8(6.16g, 7.3mmol) synthesized in example 8 and Ac-D-cys (acm) -D-Ala-SPh (2.79g, 7.0mmol) synthesized in example 3 were added to 20mL of acetonitrile, stirred, and 60mL of buffer solution (6.0M Gn · HCl,0.2M Na2HPO4, pH 6.6) was added and the solution was stirred to clear. TCEP (17.5g, 70mmol) and TFE (7.0g, 70mmol) were added, stirred at room temperature for 4 hours and the reaction monitored by HPLC. After the reaction was complete VA-044(44mg, 0.14mmol) and tBuSH (1.26g, 14mmol) were added and stirring continued at room temperature for 4 hours, after HPLC monitoring the reaction was complete, purification by reverse phase HPLC, lyophilization gave 6.12g of a white solid with a purity of 99.8% (see FIG. 5), yield 87.4%, MS [ M + H ]:1000 (see FIG. 6).

Example 10: synthesis of Etecalacetide

Compound 9(6.12g, 6.12mmol) obtained in example 9 and L-Cys-OH (3.7g,30.6mmol) were dissolved in 60mL of a mixed solvent (purified water: methanol ═ 50:10), and the pH was adjusted to 3.0 with glacial acetic acid. Further, iodine (1.6g, 12.3mmol) was added thereto, and the mixture was stirred at room temperature for 3 hours. The reaction was monitored by HPLC. Purification by reverse phase HPLC preparative after completion of the reaction gave 6.20g of a white solid with a purity of 99.9% (see FIG. 7), a yield of 96.5%, MS [ M + H ]:1049 (see FIG. 8).

Comparative examples of the liquid phase Process of the original research

EXAMPLE 11 Synthesis of Compound Z-D-Ala-D-Orn (Boc) -OMe (Compound 1b)

H-D-Orn (Boc) -OMe (11.0g) was weighed into anhydrous tetrahydrofuran and stirred to clear, cooled to 0 ℃ on an ice bath. Z-D-Ala-NCA (11.2g,47.3mmol) was added under nitrogen and stirred at room temperature for 2 hours. TLC monitored the reaction. Adding silicon-NH after the reaction₂The solid filler (5g) was stirred at room temperature for 16 hours, filtered to remove the filler, and the filtrate was concentrated and purified by 200-mesh 300-mesh silica gel column chromatography eluting with 2% methanol dichloromethane. Purification gave Compound 1b (11.9g, 66.5% yield)

EXAMPLE 12 Synthesis of the Compound H-D-Ala-D-Orn (Boc) -OMe (Compound 2b)

Compound 1b (11.9g) was added to anhydrous tetrahydrofuran and dissolved with stirring. Pb/C (6g, 10% activated carbon) was added under nitrogen, and the mixture was stirred at 40psi atmospheric pressure for 2 hours at room temperature. Filtration and concentration of the filtrate gave 8.0g of crude compound 2 b.

EXAMPLE 13 Synthesis of Compound Z-D-Orn (Boc) -D-Ala-D-Orn (Boc) -OMe (Compound 3b)

Compound 2b (8.0g) was added to anhydrous tetrahydrofuran and stirred to dissolve. The mixture was cooled to 0 ℃ in an ice bath. Z-D-Orn (Boc) -NCA (11.0g,27.7mmol) was added under nitrogen, and the mixture was stirred at room temperature for 2 hours. TLC monitored the reaction. Adding silicon-NH after the reaction₂The solid filler (15g) was stirred at room temperature for 16 hours, and then filtered to remove the filler, thereby obtaining compound 3b (9.5g, yield 55.0%) after recrystallization.

EXAMPLE 14 Synthesis of the Compound H-D-Orn (Boc) -D-Ala-D-Orn (Boc) -OMe (Compound 4b)

Compound 3b (9.5g) was added to anhydrous tetrahydrofuran and stirred to dissolve. Pb/C (1g, 10% activated carbon) was added under nitrogen, and stirred at 40psi atmospheric pressure for 2 hours at room temperature. Filtration and concentration of the filtrate gave 6.9g of crude compound 4 b.

EXAMPLE 15 Synthesis of the Compound Z-D-Orn (Boc) -D-Ala-D-Orn (Boc) -OMe (Compound 5b)

Compound 4b (6.9g) was added to anhydrous tetrahydrofuran and stirred to dissolve. Cooled to 0 ℃ in an ice bath. Z-D-Orn (Boc) -NCA (5.7g,14.5mmol) was added under nitrogen, and the mixture was stirred at room temperature for 2 hours. TLC monitored the reaction. Adding silica-NH after the reaction₂The solid filler (15g) was stirred at room temperature for 16 hours, and then filtered to remove the filler, thereby obtaining compound 5b (10.2g, yield 81.0%) after recrystallization.

EXAMPLE 16 Synthesis of the Compound H-D-Orn (Boc) -D-Ala-D-Orn (Boc) -OMe (Compound 6b)

Compound 5b (10.2g) was added to anhydrous tetrahydrofuran and dissolved with stirring. Pb/C (1g, 10% activated carbon) was added under nitrogen, and stirred at 40psi atmospheric pressure for 2 hours at room temperature. Filtration and concentration of the filtrate gave 8.0g of crude compound 6 b.

EXAMPLE 17 Synthesis of the Compound Ac-D-Cys (Acm) -D-Ala-D-Orn (Boc) -OMe (Compound 7b)

Ac-D-Cys (Acm) -OH (6.8g,29.1mmol) and compound 2b (11.9g) were weighed into anhydrous tetrahydrofuran and stirred to dissolve. Cooled to 0 ℃ in an ice bath. EDC (6.12g,31.9mmol), HOBT (4.0g,29.3mmol) and DIEPA (5.4mL,31.9mmol) were added under nitrogen and reacted at room temperature for 16 hours. The reaction solution was added to cold water, filtered, and the filter cake was washed with cold water. The filter cake was passed through a 200-mesh 300-mesh silica gel column eluting with 3% methanol in dichloromethane. After purification, compound 7b (8.3g, yield 66.0%) was obtained.

EXAMPLE 18 Synthesis of the Compound Ac-D-Cys (Acm) -D-Ala-D-Orn (Boc) -OH (Compound 8b)

Adding the compound 7b (8.3g) into a tetrahydrofuran water mixed solvent (volume ratio is 1:1), cooling to 0 ℃ in an ice bath, slowly adding 1N lithium hydroxide aqueous solution, controlling the temperature to be not more than 5 ℃, and continuously stirring at the same temperature for 4 hours. The reaction was monitored by TLC, after completion of the reaction, the pH was adjusted to 3 with citric acid, extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, passed through a column with 200-mesh 300-mesh silica gel and eluted with 5% methanol in dichloromethane. After purification, compound 8b (4.8g, 59.0% yield) was obtained.

EXAMPLE 19 Synthesis of the Compound Ac-D-Cys (Acm) -D-Ala-D-Orn (Boc) -OH (Compound 9b)

Adding the compound 7b (8.3g) into a tetrahydrofuran-water mixed solvent (volume ratio is 1:1), cooling to 0 ℃ in an ice bath, slowly adding a 1N lithium hydroxide aqueous solution, controlling the temperature to be not more than 5 ℃, and continuously stirring at the same temperature for 4 hours. The reaction was monitored by TLC, after completion of the reaction, the pH was adjusted to 3 with citric acid, extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, passed through a column with 200-mesh 300-mesh silica gel and eluted with 5% methanol in dichloromethane. After purification, compound 9b (4.8g, yield 59.0%) was obtained.

EXAMPLE 20 Compound Ac-D-Cys

Synthesis of (Acm) -D-Ala-D-Orn (Boc) -D-Ala-D-Orn (Boc) -OMe (Compound 10b)

Compound 9b (3.5g) and compound 6b (6.5g) were weighed into NMP solvent and stirred to dissolve. Cooling to 0 ℃ by using an ice bath. EDC (1.55g,8.1mmol), HONb (1.2g,6.74mmol) and DIEPA (1.4mL,8.1mmol) were added under nitrogen and reacted at room temperature for 16 hours. The reaction solution was poured into cold water, filtered, and the filter cake was washed with cold water. The filter cake was passed through a 200-mesh 300-mesh silica gel column eluting with 6% methanol in dichloromethane. Purification gave compound 10b (5.1g, yield 60.0%)

EXAMPLE 21 Compound Ac-D-Cys (Acm) -D-Ala-D-Orn (Boc) -D-Ala-D-Orn (Boc) -NH₂Synthesis of (Compound 11b)

Compound 10b (5.1g) was dissolved in methanol, followed by 48 hours of passage of ammonia gas, concentration of the reaction solution, and the residue was washed with diethyl ether, then passed through a column of 60-120 mesh silica gel, and eluted with 7% methanol in dichloromethane. Purification gave Compound 11b (3.6g, yield 72.0%)

EXAMPLE 22 the compound Ac-D-Cys (Acm) -D-Ala-D-Orn-D-Orn-D-Orn-D-Ala-D-Orn-NH₂Synthesis of (Compound 12b)

Compound 11b (3.6g) was added to 0 ℃ 5N HCl/EtOAc solvent, followed by 3mL triisopropylsilane and the reaction was continued at 0 ℃ for 1 hour. After concentration the residue was washed with diethyl ether. Compound 12b (2.2g) was obtained.

The compound Ac-D-Cys (Acm) -D-Ala-D-Arg (Boc) of example 23₂-D-Arg(Boc)₂-D- Arg(Boc)₂-D-Ala-D-Arg(Boc)₂-NH₂Synthesis of (Compound 13b)

Compound 12b (2.2g) was added to 20mL of methanol and 5mL of water, and N, N' -di-BOC-1H-1-guanidinopyrazole (5.0g,16mmol) and DIEPA (9.0mL,52.4mmol) were added thereto, followed by stirring at room temperature for 12 hours. After completion of the reaction, concentration was carried out, the residue was dissolved in ethyl acetate, washed successively with water and saturated sodium chloride, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The column was passed through 200-mesh 300-mesh silica gel, and eluted with a 70% ethyl acetate-petroleum ether mixed solvent. Purification gave compound 13b (1.6g, yield 31.0%)

EXAMPLE 24 Synthesis of Etecalacetide

Compound 13b (1.6g) was dissolved in 50% trifluoroacetic acid in dichloromethane, concentrated and precipitated with diethyl ether. And (5) drying by nitrogen. The solid and L-Cys-OH (150mg) were dissolved in 12mL of a mixed solvent (purified water: methanol 10:2), and pH was adjusted to 3.0 with glacial acetic acid. Further, iodine (220mg) was added thereto, and the mixture was stirred at room temperature for 3 hours. The reaction was monitored by HPLC. After the reaction was complete, preparative purification by reverse phase HPLC gave 1.3g of a white solid with a purity of 99.5% (see FIG. 9) and a yield of 95.5% MS [ M + H ]:1049 (see FIG. 10).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

The synthesis method of the Etelcalcetide is characterized in that the method comprises the steps of taking beta-S (Trt) -D-Arg (Boc)3-OH as a raw material, replacing alanine in a peptide sequence with cysteine, and performing coupling reaction by adopting a natural chemical connection method to prepare the Etelcalcetide; the method comprises the following steps:

step 1: with beta-S (Trt) -D-Arg (Boc)₃-OH and thiophenol (PhSH) as starting materials inObtaining a compound 1 through coupling reaction in the presence of a solvent;

step 2: Ac-D-Cys (Acm) -OH and HONb are used as raw materials, activated by a coupling agent in the presence of a solvent, and subjected to dehydration condensation with H-Ala-OH to obtain a compound 2;

and step 3: taking a compound 2 and thiophenol (PhSH) as raw materials, and carrying out condensation reaction in the presence of a solvent and a coupling agent to obtain a compound 3;

and 4, step 4: Boc-D-Cys (Trt) -OH, H-D-Arg (Pbf) -NH₂HCI is taken as a raw material, and a dipeptide fragment, namely a compound 4, is obtained through condensation reaction in the presence of a solvent and a coupling agent;

and 5: performing deprotection reaction on the compound 4 in the presence of a lysate to obtain a compound 5;

step 6: cracking a compound 5 and a compound 1 serving as raw materials in the presence of a solvent, a buffer solution, TCEP and trifluoroethanol through a natural chemical ligation reaction to obtain a compound 6;

and 7: cracking a compound 6 and a compound 1 serving as raw materials in the presence of a solvent, a buffer solution, TCEP and trifluoroethanol through a natural chemical ligation reaction to obtain a compound 7;

and 8: cracking a compound 7 and a compound 1 serving as raw materials to obtain a compound 8 through natural chemical ligation reaction in the presence of a solvent, a buffer solution, TCEP and trifluoroethanol;

and step 9: taking a compound 8 and a compound 3 as raw materials, carrying out natural chemical linking reaction in the presence of a solvent, a buffer solution, TCEP and trifluoroethanol, and then carrying out desulfurization reaction and purification in the presence of an initiator and tBuSH to obtain a compound 9;

step 10: the compound 9 and L-Cys-OH are used as raw materials, and Etecalacetide is obtained by iodine oxidation and purification.
2. The method of claim 1, wherein the solvent in step 1 comprises one or more of tetrahydrofuran, dimethylsulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, and dichloromethane;

step 1 beta-S (Trt) -D-Arg (Boc)₃The molar ratio of-OH to thiophenol (PhSH) is 1: 1.0-1.5;

the temperature of the coupling reaction in the step 1 is 20-30 ℃; the coupling reaction time is 2-10 h; the coupling agent adopted by the coupling reaction comprises a composition of DIC and a compound A and a composition of DIPEA, the compound A and a compound B, wherein the compound A is HOAt or HOBt, and the compound B is one or a composition of more than two of PyAOP, PyBOP, HATU, HBTU, TBTU or EDC.
3. The synthesis method according to claim 1 or 2, wherein the molar ratio of Ac-D-Cys (Acm) -OH to HONb in step 2 is 1: 1.0-1.5;

the solvent selected for activation in the step 2 comprises one or a composition of more than two of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform and dichloromethane;

the activation temperature in the step 2 is 20-30 ℃, and the activation time is 1-5 h;

the coupling agent adopted for activation in the step 2 is DIC or a composition of DIPEA and a compound B, and the compound B is one or a composition of more than two of PyAOP, PyBOP, HATU, HBTU, TBTU or EDC;

the solvent selected for dehydration condensation in the step 2 comprises one or a mixture of more than two of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane and water;

the temperature of the dehydration condensation in the step 2 is 20-30 ℃, and the time of the dehydration condensation is 1-3 h.
4. The synthesis method according to claim 1 or 2, wherein the molar ratio of compound 2 to thiophenol (PhSH) in step 3 is 1:1.0 to 1.5;

in step 3, the solvent used in the condensation reaction comprises one or a combination of more than two of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform and dichloromethane;

in the step 3, the temperature of the condensation reaction is 20-30 ℃, and the time of the condensation reaction is 2-10 h;

in the step 3, the coupling agent is a composition of DIC and a compound A, and a composition of DIPEA, a compound A and a compound B, wherein the compound A is HOAt or HOBt, and the compound B is one or a mixture of more than two of PyAOP, PyBOP, HATU, HBTU, TBTU or EDC.
5. The method of claim 1 or 2, wherein in step 4 Boc-D-Cys (Trt) -OH and H-D-Arg (Pbf) -NH₂The molar ratio of HCI is 1: 1.0-1.5;

the temperature of the condensation reaction in the step 4 is 20-30 ℃, and the time of the condensation reaction is 2-10 h;

the solvent in the step 4 comprises one or a mixture of more than two of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform and dichloromethane;

in the step 4, the coupling agent is a composition of DIC and a compound A, and a composition of DIPEA, a compound A and a compound B, wherein the compound A is HOAt or HOBt, and the compound B is one or a mixture of more than two of PyAOP, PyBOP, HATU, HBTU, TBTU or EDC.
6. The method of claim 1 or 2, wherein the lysing solution in step 5 is TFA, H₂A combination of O and TIS; TFA, H₂The volume ratio of O to TIS is 95:2.5: 2.5;

the temperature of the protecting group removing reaction in the step 5 is 20-30 ℃, and the time of the protecting group removing reaction is 2-4 h.
7. The synthesis method according to claim 1 or 2, wherein in the step 6 to 9, the molar ratio of the compound 5, the compound 6, the compound 7, the compound 8 to the compound 1 or the compound 3 is 1.0 to 1.1: 1;

in the steps 6-9, the buffer solution is 6.0M guanidine hydrochloride (Gn & HCl) and 0.2M Na₂HPO₄The pH value of the mixed solution is 5.6-7.6;

in the step 6-9, the solvent comprises one or a mixture of more than two of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform and dichloromethane;

the temperature of the natural chemical connection reaction in the step 6-9 is 20-30 ℃, and the time of the natural chemical connection reaction is 2-6 h;

in the step 6-9, the consumption of TCEP and trifluoroethanol is 5-15 equivalents of the raw materials;

the cracking solution adopted in the cracking in the step 6-8 is TFA and H₂O, TIS composition, TFA, H₂O, TIS in a volume ratio of 95:2.5: 2.5;

the temperature of the pyrolysis in the steps 6-8 is 20-30 ℃, and the time of the pyrolysis is 2-4 h.
8. The synthetic method of claim 1 or 2 wherein in step 9 the initiator is VA-044; the using amount of the initiator is 0.01-0.03 equivalent of that of the compound 3; the dosage of tBuSH is 1-3 equivalents of the compound 3;

the temperature of the desulfurization reaction in the step 9 is 20-30 ℃, and the time of the desulfurization reaction is 2-4 h;

the purification in step 9 is performed by reversed phase high pressure liquid chromatography comprising: using reverse-phase octadecylsilane as stationary phase, using 0.1% trifluoroacetic acid water solution/acetonitrile as mobile phase, collecting maximum peak fraction, concentrating under reduced pressure at 30 deg.C to 1/4 volume, and lyophilizing.
9. The method of claim 1 or 2, wherein the solvent used in the iodine oxidation in step 10 is one or a mixture of more than two of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane, methanol, ethanol and water;

in the step 10, adjusting the pH value of iodine oxidation to 2-4 by adopting glacial acetic acid;

in the step 10, the amount of the iodine is 1-3 equivalent of the raw material;

in the step 10, the temperature of iodine oxidation is 20-30 ℃, and the time of iodine oxidation is 1-5 h;

in the step 10, the purification adopts reversed phase high pressure liquid chromatography; the reversed phase high pressure liquid chromatography comprises: using reverse octadecylsilane as stationary phase, using 0.1% trifluoroacetic acid water solution/acetonitrile as mobile phase, collecting maximum peak fraction, concentrating under reduced pressure at 30 deg.C to 1/4 volume, and lyophilizing.