A method for preparing an etelcalcetide

Abstract

The present application relates to a preparation method of etelcalcetide, and belongs to the technical field of biotechnology. The present application effectively reduces the generation of one [Ac-D-Cys] 1 impurities (the impurities are very close to the main peak retention time in HPLC, and greatly affect the purification efficiency) due to the removal of acetyl by hydrolysis; in addition, the present application first carries out the sulfhydryl modification of one [Ac-D-Cys] 1 , then removes the Pbf protecting group, and monitors the reaction end point through Ellman reagent, so that the generation of sulfonated impurities can be reduced. In addition, the present application uses different solvents for multiple washing, which can effectively control the generation of homodimers to improve the product yield. In summary, the preparation method of the present application effectively reduces the generation of acetylated impurities, sulfonated impurities and homodimers, reduces the difficulty of purifying impurities, and has important application value in industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and particularly to a preparation method of etelcalcetide. BACKGROUND

[0002] Etelcalcetide is mainly used for treating secondary hyperparathyroidism (SHPT). For those adult chronic kidney disease (CKD) patients receiving hemodialysis treatment, etelcalcetide can inhibit the synthesis of PTH by regulating the calcium-sensitive receptors of parathyroid glands and improving the sensitivity of extracellular calcium ions, and can also up-regulate the transcription and translation of parathyroid calcium-sensitive receptor mRNA and vitamin D receptor mRNA to inhibit the synthesis of PTH.

[0003] The peptide sequence of etelcalcetide is: Ac-D-Cys 1 -D-Ala 2 -D-Arg 3 -D-Arg 4 -D-Arg 5 -D-Ala 6 -D-Arg 7 -NH2. The side chain sulfhydryl group of D-Cys1 in the main chain is connected to the side chain sulfhydryl group of L-Cys in the form of a disulfide bond.

[0004] The preparation method disclosed in the patent with publication number CN115490756A adds the peptide resin and H-Cys(S-Py)-OH together into a high-concentration TFA cleavage solution for reaction, and the side chain protection group pbf of D-Arg will be cleaved to form a positive ion, which is easily captured by the sulfhydryl group of D-Cys 1 , forming a D-Cys 1 sulfonated impurity. Moreover, the exposed amino group of the main chain peptide resin after cleavage also has a potential risk of reaction with H-Cys(S-Py)-OH.

[0005] The preparation method disclosed in the patent with publication number CN108218957A removes the side chain protection group of D-Cys 1 1 and cuts the resin by using dilute acid, and because a long reaction time is required, part of the side chain protection group pbf of D-Arg will be cleaved, also forming a D-Cys 1 sulfonated impurity. Similarly, the exposed amino group after cutting the peptide resin also has a potential risk of reaction with H-Cys(S-Py)-OH.

[0006] In the preparation method disclosed in the patent with publication number CN106795201A, [D-Cys(S-Py)] 1The side chain is not stable under strong acid, and there is a risk of breaking and removing, which makes the reaction unable to proceed and affects the yield. At the same time, the positive ion reaction formed by the side chain protection group pbf of D-Arg which is also cleaved off produces a series of impurities.

[0007] In summary, the several solid-liquid combined preparation methods of eptifibatide disclosed at present can form acetylated impurities and sulfonated impurities which are difficult to separate and have low yield, and therefore a preparation method of eptifibatide with high yield and easily separated impurities is urgently needed. SUMMARY

[0008] To this end, the technical problem to be solved by the present application is to overcome the problems of low yield and difficult separation of impurities in the preparation method of eptifibatide in the prior art.

[0009] To solve the above technical problems, the present application provides a preparation method of eptifibatide, which uses an amino resin as a solid phase carrier to prepare a main chain peptide resin by stepwise coupling: Ac-D-Cys(Mmt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-resin. Then remove the Mmt protection of the side chain of one D-Cys 1 in the solid phase system, add Boc-Cys(S-Py)-OH to obtain an eptifibatide peptide resin. Through steps such as cleavage, purification, salt conversion and freeze-drying, eptifibatide is obtained. Specifically, the side chain protection group pbf of D-Arg will be cleaved to form a positive ion, which is easily captured by the thiol group of D-Cys 1 to form sulfonated impurities. To solve this problem, the present application first removes the Mmt protection of the side chain of D-Cys 1 and connects Boc-Cys(S-Py)-OH to avoid exposure of the thiol group of D-Cys 1 , and then removes the side chain protection group pbf of D-Arg, thereby avoiding the generation of difficult-to-separate sulfonated impurities due to the exposure of the thiol group of one D-Cys 1 side chain and the reaction with pbf; in addition, to reduce the generation of difficult-to-separate deacetylated impurities, the present application controls the cleavage temperature to reduce the generation of difficult-to-separate deacetylated impurities; at the same time, the number of washing times with different solvents is increased to reduce the generation of homodimer impurities. Therefore, the purity of the eptifibatide of the present application reaches 90.66% and does not produce difficult-to-separate deacetylated impurities, sulfonated impurities and homodimer impurities.

[0010] The first object of the present application is to provide a preparation method of eptifibatide, comprising the following steps:

[0011] S1, mixing Fmoc-D-Ala-OH, Fmoc-D-Arg(R)-OH and Fmoc-D-Cys(X)-OH with a deprotection agent respectively to obtain D-Ala-OH, D-Arg(R)-OH and D-Cys(X)-OH which are removed Fmoc protection, and coupling D-Ala-OH, D-Arg(R)-OH and D-Cys(X)-OH to an amino resin according to the peptide sequence from C-terminal to N-terminal of the eticapeptide main chain in the presence of a condensing agent, to obtain a first eticapeptide resin, wherein X is selected from Trt, Mmt or tBu protecting group, and R includes a Pbf protecting group;

[0012] S2, adding a mixed solution of trifluoroacetic acid and dichloromethane to the first eticapeptide resin for deprotection treatment and washing to obtain a second eticapeptide resin;

[0013] S3, adding Boc-Cys(S-Py)-OH to the second eticapeptide resin prepared in S2 in the presence of a catalyst to obtain a thiolated third eticapeptide resin;

[0014] S4, cleaving the third eticapeptide resin to obtain an eticapeptide after purification, wherein the R and the amino resin are removed.

[0015] Further, the temperature of the cleavage reaction in step S4 is 15-25°C.

[0016] Further, the deprotection agent is a mixed solution of piperidine and DMF.

[0017] Further, Fmoc-D-Cys(X)-OH is preferably Fmoc-D-Cys(Mmt)-OH (i.e. X is Mmt protecting group) which has a relatively weak acid resistance of side chain.

[0018] Further, the amino resin includes Rink Amide-AM Resin, wherein the resin substitution degree is 0.5 mmol / g.

[0019] Further, in step S1, D-Cys(X)-OH, D-Ala-OH, D-Arg(R)-OH, D-Arg(R)-OH, D-Arg(R)-OH and D-Ala-OH are sequentially coupled to the amino resin in the presence of a condensing agent and a racemization protection agent to obtain the eticapeptide resin.

[0020] Further, the condensing agent is selected from one or more of dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the racemic protecting agent is selected from one or more of 1-hydroxybenzotriazole (HOBt), 4-dimethylaminopyridine, and benzotriazole-1-yloxotris(dimethylamino)phosphoryltriamine. DCC (dicyclohexylcarbodiimide) readily forms an O-isourea intermediate during the reaction, which is readily converted to racemic oxazolone. To prevent this, HOBt (1-hydroxybenzotriazole) can be added to react with it, forming another intermediate with a faster aminolysis rate, thereby effectively inhibiting the formation of the oxazolone active intermediate.

[0021] DMAP (4-dimethylaminopyridine) is a commonly used catalyst that accelerates amide bond formation while reducing racemization. It is often used in conjunction with DCC to form the DCC / DMAP system, which is widely applied in peptide synthesis and other organic synthesis.

[0022] TBTU (benzotriazol-1-yloxotris(dimethylamino)phosphoryltriamine) is a highly efficient condensation reagent that promotes amide bond formation under mild conditions with minimal racemic effect. It is typically used in conjunction with DIPEA (diisopropylethylamine) to form the TBTU / DIPEA system, suitable for the condensation reactions of various amino acids.

[0023] Furthermore, the first etacaptide resin undergoes acetylation treatment, which involves adding acetic anhydride and pyridine to the first etacaptide resin. The main reason for acetylation treatment of the etacaptide resin is to block the N-terminus of the peptide, preventing its degradation and enhancing its stability.

[0024] Further, in step S2, the deprotection treatment involves adding a mixed solution of trifluoroacetic acid as a solute and dichloromethane as a solvent to the first etkatide resin for reaction, wherein the concentration of the mixed solution is 5%-20%, the deprotection treatment is performed 4-5 times, and the time for each deprotection treatment is 4-5 minutes.

[0025] Further, in step S2, the washing involves adding DCM solution to the first etkatide resin after deprotection treatment and washing it three times, then adding a mixed solution of 10%-20% N,N-diisopropylethylamine and dichloromethane and washing for 1-2 minutes, repeating 2-5 times, and finally adding N,N-dimethylformamide to wash the resin 3-6 times.

[0026] Further, in step S2, the reaction progress is monitored using Ellman's reagent during the deprotection process. The deprotection process ends when Ellman's reagent changes color. This process involves the removal of one D-Cys atom.1 The reaction endpoint was monitored by detecting thiol exposure using Ellman's reagent before and after Mmt protection of the side chain, and before and after docking with Boc-Cys(S-Py)-OH. Using Ellman's reagent is more efficient than commonly used HPLC methods. This invention utilizes a single D-Cys... 1 The variation in the presence or absence of exposed thiol groups on the side chain, and the introduction of a specific amino acid into the side chain, allows the reaction endpoint to be indicated by Ellman's reagent colorimetric reaction during solid-phase reaction control, without relying on HPLC, thus simplifying the detection of the reaction endpoint.

[0027] Furthermore, the catalyst described in step S3 includes DIPEA.

[0028] Further, the cleavage reaction in step S4 involves adding a cleavage buffer to itcatide, wherein the cleavage buffer is a mixture of trifluoroacetic acid, triisopropylsilane, and water, with a volume ratio of (90-96):(2-5):(2-5). The cleavage buffer contains trifluoroacetic acid, which can remove the Pbf protecting group from the peptide chain.

[0029] The beneficial effects of this invention are:

[0030] This invention effectively reduces the number of [Ac-D-Cys] sites by adjusting the pyrolysis temperature. 1 Impurities generated by the removal of acetyl groups due to hydrolysis (these impurities have retention times very close to the main peak in HPLC, significantly affecting purification efficiency); in addition, this invention first performs a [Ac-D-Cys] single-phase [Ac-D-Cys] reaction. 1 The product undergoes thiol modification followed by Pbf protecting group removal to reduce the formation of sulfonated impurities. Furthermore, the invention utilizes multiple washes with different solvents to effectively control the formation of homodimers and improve product yield. Therefore, this invention effectively reduces the formation of acetylated impurities, sulfonated impurities, and homodimers, lowering the difficulty of impurity purification and possessing significant application value in industrial production. Attached Figure Description

[0031] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0032] Figure 1 This is the main flowchart of the present invention;

[0033] Figure 2 This is the HPLC chromatogram of the crude itcatide peptide obtained in Example 2;

[0034] Figure 3 This is the HPLC chromatogram of the crude itcatide peptide obtained in Example 3;

[0035] Figure 4 This is the HPLC chromatogram of crude itcatide obtained in Comparative Example 1.

[0036] Figure 5 This is the HPLC chromatogram of the crude itcatide peptide obtained in Comparative Example 2. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0038] Example 1: Synthesis of itcatide resin

[0039] (1) Preparation of deprotected amino resin: Weigh 384.6 g (200 mmol, degree of substitution 0.52 mmol / g) of Rink Amide AM resin, add DMF and stir to swell for 30 min, then remove Fmoc protection with 20% piperidine / DMF solution. After deprotection is complete, remove the liquid and wash 5 times with DMF. Ninhydrin detection (Kaiser Test): the resin is blue, indicating complete deprotection.

[0040] (2) Preparation of deprotected amino acids: Weigh Fmco-D-Arg(pbf)-OH (3 eq, 389.3 g) and HOBt (3.6 eq, 97.3 g), dissolve them in 3 L of DMF, then add DIC (3.6 eq, 111.3 mL), stir to activate, and then add to the deprotected amino resin. Stir to react. During the reaction, a small amount of resin was tested for ninhydrin; the resin was transparent and colorless, indicating that the condensation reaction was complete. Wash the resin three times with DMF. Remove Fmoc protection with 20% piperidine / DMF solution (the deprotection treatment of Fmoc-D-Ala-OH and Fmoc-D-Cys(Mmt)-OH is similar to that of Fmco-D-Arg(pbf)-OH). After deprotection is complete, remove the liquid and wash five times with DMF. Ninhydrin test showed that the resin was blue, indicating complete deprotection.

[0041] (3) Peptide chain condensation: Fmoc-D-Ala-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Ala-OH, Fmoc-D-Cys(Mmt)-OH are condensed sequentially from the C-terminus to the N-terminus of the peptide sequence, and Fmoc protection is removed.

[0042] (4) Acetylation of the first itca peptide resin: Using 3L DMF as solvent, acetic anhydride (5 eq, 94.0 ml) and pyridine (5 eq, 80.7 ml) were added and mixed, then added to the peptide resin and reacted at room temperature for 30 minutes. The resin was washed 5 times with DMF. A sample was taken for ninhydrin detection; the resin was colorless and transparent. The resin was washed 3 times with 3L anhydrous methanol for 10 minutes each time. The resin was vacuum dried at room temperature for 15 hours to obtain 871.9 g of the main chain peptide resin.

[0043] Example 2: Synthesis of itcatide

[0044] Weigh 21.8 g (5 mmol) of the main-chain peptide resin obtained in Example 1. Add DMF and stir to swell for 30 min. Prepare 100 mL of 10% trifluoroacetic acid / DCM solution and add it to the peptide resin. Stir for 5 min, filter off the reaction solution, and repeat the operation 5 times. Wash with DCM 3 times. Prepare 10% DIPEA / DCM solution and wash the resin twice, 2 min each time. Wash with DMF 5 times. Take a small amount of resin and add Ellman's reagent for detection; the resin turns purple-red. Dissolve Boc-Cys(S-Py)-OH (3 eq, 5.0 g) in 100 mL of DMF, then add 5.2 mL of DIPEA (6 eq). Stir and add to the peptide resin for reaction. During the reaction, take a small amount of resin and add Ellman's reagent for detection; the resin becomes transparent and colorless, which is the reaction endpoint. Wash with DMF 5 times, wash with anhydrous methanol 3 times, and then vacuum dry to obtain 21.6 g of itka peptide resin.

[0045] Prepare 172.8 mL of lysis buffer (TFA:TIS:water = 95:2.5:2.5). The volume of the lysis buffer is 8 times the weight of the resin (8 mL / g). After cooling to 2-8℃, add it to the peptide resin and stir for 3 h, maintaining the temperature at 25℃. Filter, add the filtrate to 1036.8 mL of methyl tert-butyl ether (6 mL / mL lysis buffer, around 0℃), centrifuge, wash 3 times with methyl tert-butyl ether, and vacuum dry the wet product at room temperature for 12 h. Weigh to obtain 5.2 g of crude peptide. HPLC analysis shows a purity of 90.66% and 1.15% homodimer. The HPLC chromatogram is shown below. Figure 2 As shown.

[0046] Example 3

[0047] Weigh 21.8 g (5 mmol) of the main-chain peptide resin obtained in Example 1. Add DMF and stir to swell for 30 min. Prepare 100 mL of 10% trifluoroacetic acid / DCM solution and add it to the peptide resin. Stir for 5 min, filter off the reaction solution, and repeat the operation 5 times. Wash with DCM 3 times. Prepare 10% DIPEA / DCM solution and wash the resin twice, 2 min each time. Wash with DMF 5 times. Take a small amount of resin and add Ellman's reagent for detection; the resin turns purple-red. Dissolve Boc-Cys(S-Py)-OH (3 eq, 5.0 g) in 100 mL of DMF, then add 5.2 mL of DIPEA (6 eq). Stir and add to the peptide resin for reaction. During the reaction, take a small amount of resin and add Ellman's reagent for detection; the resin becomes transparent and colorless, which is the reaction endpoint. Wash with DMF 5 times, wash with anhydrous methanol 3 times, and then vacuum dry to obtain 21.6 g of itka peptide resin.

[0048] Prepare 172.8 mL of lysis buffer (TFA:TIS:water = 95:2.5:2.5). The volume of the lysis buffer is 8 times the weight of the resin (8 mL / g). After cooling to 2-8℃, add it to the peptide resin and stir for 3 h, controlling the temperature at 15℃. Filter, add the filtrate to 1036.8 mL of methyl tert-butyl ether (6 mL / mL lysis buffer, around 0℃), centrifuge, wash 3 times with methyl tert-butyl ether, and vacuum dry the wet product at room temperature for 12 h. Weigh 5.2 g of crude peptide. HPLC analysis showed a purity of 65.33% and 1.26% homodimer. At lower lysis temperatures, dimer impurities are less likely to form, but a series of peaks with different amino acid side chain protecting groups are generated. These impurities are far from the main peak of ittca peptide, making them easier to separate. The HPLC chromatogram is shown below. Figure 3 As shown.

[0049] Comparative Example 1

[0050] Weigh 21.8 g (5 mmol) of the main-chain peptide resin obtained in Example 1. Add DMF and stir to swell for 30 min. Prepare 100 ml of 10% trifluoroacetic acid / DCM solution, add it to the peptide resin, stir for 5 min, filter off the reaction solution, and repeat the operation 5 times. Wash 5 times with DMF. Take a small amount of resin and add Ellman's reagent for detection; the resin turns purple-red. Dissolve Boc-Cys(S-Py)-OH (3 eq, 5.0 g) in 100 ml of DMF, then add 5.2 ml of DIPEA (6 eq). Stir and add to the peptide resin for reaction. During the reaction, take a small amount of resin and add Ellman's reagent for detection; the resin becomes transparent and colorless, which is the reaction endpoint. Wash 5 times with DMF, wash 3 times with anhydrous methanol, and then vacuum dry to obtain 19.8 g of Itka peptide resin.

[0051] Prepare 158.4 ml of lysis buffer (TFA:TIS:water = 95:2.5:2.5). The volume of the lysis buffer is 8 times the weight of the resin (8 ml / g). After cooling to 2-8℃, add it to the peptide resin and stir for 3 hours, maintaining the temperature at 25℃. Filter, add the filtrate to 950.4 ml of methyl tert-butyl ether (6 ml / ml lysis buffer, around 0℃), centrifuge, wash 3 times with methyl tert-butyl ether, and dry the wet product under vacuum at room temperature for 12 hours. Weigh to obtain 4.8 g of crude peptide. HPLC analysis shows a purity of 63.94% and a homodimer content of 6.65%. The HPLC chromatogram is shown below. Figure 4 As shown.

[0052] Example 2 involved multiple washes with DCM and 10% DIPEA / DCM after Mmt protection removal. The crude peptide purities were 81.74% and 63.94%, respectively. The purity of Example 2 was significantly higher than that of Comparative Example 1. The homodimer impurities in the two experiments were 2.39% and 6.65%, respectively, showing a significant reduction in Example 2.

[0053] Comparative Example 2

[0054] Weigh 21.8 g (5 mmol) of the main-chain peptide resin obtained in Example 1. Add DMF and stir to swell for 30 min. Prepare 100 mL of 10% trifluoroacetic acid / DCM solution and add it to the peptide resin. Stir and react for 5 min, filter off the reaction solution, and repeat the operation 5 times. Wash with DCM 3 times. Prepare 10% DIPEA / DCM solution and wash the resin twice, 2 min each time. Wash with DMF 5 times. Take a small amount of resin and add Ellman's reagent for detection; the resin turns purple-red. Dissolve Boc-Cys(S-Py)-OH (3 eq, 5.0 g) in 100 mL of DMF, then add 5.2 mL of DIPEA (6 eq). Stir and add to the peptide resin for reaction. During the reaction, take a small amount of resin and add Ellman's reagent for detection; the resin becomes transparent and colorless, which is the reaction endpoint. Wash with DMF 5 times, wash with anhydrous methanol 3 times, and then vacuum dry to obtain 20.5 g of itka peptide resin.

[0055] Prepare 164.0 mL of lysis buffer (TFA:TIS:water = 95:2.5:2.5). The volume of the lysis buffer is 8 times the weight of the resin (8 mL / g). After cooling to 2-8℃, add it to the peptide resin and stir for 3 h, maintaining the temperature at 35℃. Filter, and add the filtrate to 984 mL of methyl tert-butyl ether (6 mL / mL lysis buffer, around 0℃). Centrifuge, wash 3 times with methyl tert-butyl ether, and vacuum dry the wet product at room temperature for 12 h. Weigh to obtain 5.0 g of crude peptide. HPLC analysis shows a purity of 58.36% and deacetylation impurities of 15.02%. The HPLC chromatogram is shown below. Figure 5 As shown.

[0056] Compared to Comparative Example 2, Example 2 controlled the temperature at 25°C during the lysis process, while Comparative Example 2 reached 35°C. The crude peptide purities were 90.66% and 58.36%, respectively. The purity of Example 2 was significantly higher than that of Comparative Example 2. Comparative Example 2 produced a large amount of deacetylated impurities, accounting for 15.02%, while none were produced in Example 2.

[0057] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing iticatide, characterized in that, Includes the following steps: S1. Fmoc-D-Ala-OH, Fmoc-D-Arg(R)-OH and Fmoc-D-Cys(X)-OH are mixed with a deprotecting agent to obtain D-Ala-OH, D-Arg(R)-OH and D-Cys(X)-OH deprotected from Fmoc. Under the presence of a condensing agent, D-Ala-OH, D-Arg(R)-OH and D-Cys(X)-OH are linked to an amino resin according to the C-terminus to N-terminus peptide sequence of the ittcapeptide backbone and then acetylated to obtain the first ittcapeptide resin, wherein X is selected from Trt, Mmt or tBu protecting groups and R is a Pbf protecting group. S2. The first itcatide resin is subjected to deprotection treatment and washing to obtain the second itcatide resin from which X is removed. S3. In the presence of a catalyst, Boc-Cys(S-Py)-OH is added to the second ittcapeptide resin prepared in S2 to react and obtain the thiolized third ittcapeptide resin. S4. The third itcatide resin is subjected to a cleavage reaction, and after purification, itcatide with R and amino resin removed is obtained. The temperature of the pyrolysis reaction in step S4 is 15-25℃.

2. The preparation method according to claim 1, characterized in that, In step S1, in the presence of a condensing agent and a racemic protectant, D-Cys(X)-OH, D-Ala-OH, D-Arg(R)-OH, D-Arg(R)-OH, D-Arg(R)-OH, and D-Ala-OH are sequentially coupled to an amino resin to obtain the itcatide resin.

3. The preparation method according to claim 2, characterized in that, The condensing agent is selected from one or more of dicyclohexylcarbodiimide, diisopropylcarbodiimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the racemic protecting agent is selected from one or more of 1-hydroxybenzotriazole, 4-dimethylaminopyridine, and benzotriazole-1-yloxotris(dimethylamino)phosphoryltriamine.

4. The preparation method according to claim 1, characterized in that, The amino resin is Rink Amide-AM resin.

5. The preparation method according to claim 1, characterized in that, The acetylation treatment in step S1 involves adding acetic anhydride and pyridine to the first etacitide resin.

6. The preparation method according to claim 1, characterized in that, In step S2, the deprotection treatment involves adding a mixed solution of trifluoroacetic acid as a solute and dichloromethane as a solvent to the first etkatide resin for reaction. The concentration of the mixed solution is 5%-20%, and the deprotection treatment is performed 4-5 times, with each deprotection treatment lasting 4-5 minutes.

7. The preparation method according to claim 1, characterized in that, In step S2, the washing process involves adding dichloromethane to the first itacinide resin after deprotection treatment and washing it three times, then adding a mixed solution of 10%-20% N,N-diisopropylethylamine and dichloromethane and washing for 1-2 minutes, repeating this process 2-5 times, and finally adding N,N-dimethylformamide and washing 3-6 times.

8. The preparation method according to claim 1, characterized in that, In step S2, the reaction progress is monitored using Ellman's reagent during the deprotection process. The deprotection process ends when Ellman's reagent changes color.

9. The preparation method according to claim 1, characterized in that, The catalyst mentioned in step S3 is N,N-diisopropylethylamine.

10. The preparation method according to claim 1, characterized in that, The cleavage reaction in step S4 involves adding a cleavage solution to itcatide, wherein the cleavage solution is a mixture of trifluoroacetic acid, triisopropylsilane and water, and the volume ratio of trifluoroacetic acid, triisopropylsilane and water is (90-96):(2-5):(2-5).

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