Method for synthesizing carbetocin through double-fragment solid-liquid phase

Abstract

The invention relates to a method for synthesizing carbetocin through a double-fragment solid-liquid phase, and mainly solves the technical problems that the purity of crude peptide synthesized through a pure solid-phase long fragment in an existing synthesis method is low, and special cysteine and an expensive deprotection metal reagent are used. According to the technical scheme, the method for synthesizing the carbetocin through the double-fragment solid-liquid phase is characterized in that synthesis of the carbetocin is achieved through chemical coupling and cyclization of two fragments, and the carbetocin is obtained through double-fragment coupling at an Asn site or a Cys site and then cyclization. The synthesis yield and purity are improved, and large-scale industrial production of carbetocin is facilitated.

Description

Technical Field

[0001] The invention belongs to the field of polypeptide drug preparation, and particularly relates to a method for synthesizing carbetocin in a double-fragment solid-liquid phase. Background Art

[0002] Carbetocin is a synthetic, long-acting oxytocin cyclic nonapeptide analog with agonist properties. It is effective in preventing uterine atony and postpartum hemorrhage. It was developed by Ferring Inc. of Canada and launched in China in 2009. The clinical pharmacological properties of carbetocin are similar to those of oxytocin. Pharmacological research results show that after entering the human body, carbetocin can bind to the oxytocin body of uterine smooth muscle, induce rhythmic contraction of the uterus, and strengthen the contraction frequency and uterine tension on the basis of the original contraction of the uterus, thereby improving the symptoms of postpartum hemorrhage in parturients.

[0003] Studies have shown that, compared with oxytocin, which can only promote the contraction of the upper part of the uterus but has a poor effect on the lower part, carbetocin can have a good contraction effect on the entire uterus. In addition, carbetocin also has the characteristics of long half-life, single administration, long maintenance time, fewer side effects, and better efficacy.

[0004] Carbetocin has the following structure:

[0005] The amino acid abbreviations of carbetocin are: .

[0006] Although the existing liquid phase, solid phase and solid-liquid combined synthesis methods have achieved the synthesis of carbetocin to a certain extent, they generally have problems such as low yield, many by-products, complex operation, expensive catalysts or reagents, which seriously limit large-scale industrial production.

[0007] Chinese patent CN102477082A describes a pure liquid phase synthesis method of carbetocin, which completes the synthesis and coupling of three intermediates under liquid phase conditions, and finally obtains carbetocin through catalyst cyclization under alkaline conditions. However, this method has problems such as low yield, many by-products and complex post-processing, and is not suitable for large-scale production.

[0008] Chinese patent CN102260326A describes a solid-phase preparation method for carbetocin, which uses a new protected amino acid Fmoc-Cys ((CH2)3COOAll), overcomes the shortcomings of the prior art, and uses the same solid-phase coupling reaction as the peptide chain for cyclization. The reaction conditions are mild and the cyclization rate reaches more than 99.5%. However, when removing the All protecting group, Pd[P(C 6 H 5 ) 3 ] 4 Catalyst poisoning will occur with phenylsilane, and heavy metal substances will exist in the product that are difficult to remove, resulting in the actual yield of the product being much lower than the theoretical yield.

[0009] Chinese patent CN102796178A describes a method for preparing carbetocin, which uses a novel sulfhydryl-protected amino acid Fmoc-Cys ((CH 2 ) 3 COOCH 2 CH 2 CN) as a raw material, which is expensive, and Fmoc-Cys ((CH 2 ) 3 COOCH 2 CH 2 CN) protection has many side reactions, which is not conducive to large-scale production.

[0010] European Patent ES 2115543 describes a solid-liquid synthesis method, which mainly uses conventional solid-phase peptide synthesis to obtain linear carbetocin and then cyclizes it under liquid alkaline conditions. However, during the liquid-phase oxidation process, LiOH and NaHCO 3 and DMAP raw materials, which will lead to the generation of many impurities, reducing the purity and use effect of the product. In addition, the method is difficult to form intramolecular sulfide bonds, but disulfide bonds between molecules are very easy to form, resulting in extremely low crude product yield, which is not conducive to industrial production. Summary of the invention

[0011] The present invention aims to overcome the above problems and provide a method for synthesizing carbetocin in a double-fragment solid-liquid phase, thereby improving the synthesis yield and purity and facilitating the large-scale industrial production of carbetocin.

[0012] The technical scheme of the present invention is as follows: a method for synthesizing carbetocin in a double-fragment solid-liquid phase, characterized in that the synthesis of carbetocin is achieved by chemical coupling and cyclization of two fragments, and the method obtains carbetocin by performing double-fragment coupling at the Asn site or the Cys site and then cyclizing.

[0013] The double-fragment coupling is performed at the Asn site, followed by cyclization to obtain carbetocin, which specifically comprises the following steps: 1.1 Using 2-Cl-CTC resin as a carrier, the corresponding Fmoc-protected amino acids in the amino acid sequence of fragment I were sequentially connected through standard Fmoc solid-phase peptide synthesis, and then the chlorobutyric acid at the N-terminus was coupled using a condensation reagent. Finally, a 20% HFIP DCM solution (volume concentration) or a 1% TFA DCM solution (volume concentration) was used for cleavage to obtain a fragment I containing side chain protection, Cl-(CH 2 ) 3 CO-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-OH.

[0014] 1.2 Using Sieber resin as a carrier, the corresponding Fmoc-protected amino acids in the amino acid sequence of fragment II were sequentially connected through standard Fmoc solid-phase peptide synthesis, and then cleaved using 2-5% TFA in DCM solution (volume concentration) to obtain fragment II with a side chain protecting group and a free amino group at the N-terminus, NH 2 -Cys(Trt)-Pro-Leu-Gly-NH 2 .

[0015] 1.3 After the fragment I and the fragment II are coupled in the liquid phase, the reaction solution is precipitated with saline, washed, and dried. The obtained solid is cleaved with a cleavage solution, precipitated with cold ether, filtered, and dried to obtain a crude linear oxytocin peptide.

[0016] 1.4 The obtained linear carbetocin crude peptide is cyclized under alkaline conditions in a solvent to obtain carbetocin crude peptide, which is then purified and freeze-dried to obtain carbetocin.

[0017] The double-fragment coupling is performed at the Cys site, followed by cyclization to obtain carbetocin, which specifically comprises the following steps: 2.1 Using 2-Cl-CTC resin as a carrier, the corresponding Fmoc-protected amino acids in the amino acid sequence of fragment III were sequentially connected through standard Fmoc solid-phase peptide synthesis, and then the chlorobutyric acid at the N-terminus was coupled using a condensation reagent. Finally, 20% HFIP DCM solution (volume concentration) or 1% TFA DCM solution (volume concentration) was used for cleavage to obtain a fragment III containing side chain protection, Cl-(CH 2 ) 3 CO-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-OH.

[0018] 2.2 Using Rink Amide resin as a carrier, the corresponding Fmoc-protected amino acids in the amino acid sequence of fragment IV were sequentially connected through standard Fmoc solid-phase peptide synthesis, and fragment IV with a side chain protecting group and a free amino group at the N-terminus was obtained after cleavage using a cleavage solution. 2 -Pro-Leu-Gly-NH 2 .

[0019] 2.3 After the fragment III and the fragment IV are coupled in the liquid phase, the reaction solution is precipitated with saline, washed, and dried. The obtained solid is cleaved with a cleavage solution, precipitated with cold ether, filtered, and dried to obtain a crude linear oxytocin peptide.

[0020] 2.4 The obtained linear carbetocin crude peptide is cyclized under alkaline conditions in a solvent to obtain carbetocin crude peptide, which is then purified and freeze-dried to obtain carbetocin.

[0021] Preferably, the lysis solution used in step 1.1 is a DCM solution with a volume concentration of 20% HFIP.

[0022] Preferably, the lysis solution used in steps 1.3, 2.2 and 2.3 is TFA with a volume concentration of 90-95%.

[0023] Preferably, the condensation reagent used in the synthesis of the fragments in steps 1.1, 1.2, 2.1, and 2.2 and the coupling of the fragments in steps 1.3 and 2.3 is one of DIC, DCC, EDCI, HATU, TBTU, HCTU, HBTU, PyOxim, PyAop or PyBop, the auxiliary agent used in the condensation is one of HOAt, HOBt, Cl-HOBt, DIEA, Oxyma or NMM, the solvent used in the condensation is DMF, and the amount of each component used in the coupling is calculated by molar ratio, amino acid / chlorobutyric acid: condensation reagent: auxiliary agent = 1: 1: (1-2), fragment I / fragment III: fragment II / fragment IV: condensation reagent: auxiliary agent = 1: 1: 1: (1-2), and the preferred condensation reagent / auxiliary agent for amino acid condensation is DIC / Oxyma, amino acid: DIC: Oxyma = 1: 1.2: 1. The preferred condensation reagent / auxiliary agent for chlorobutyric acid condensation is DIC / HOBt, chlorobutyric acid: DIC: HOBt = 1: 1.2: 1. The preferred condensation reagent / auxiliary agent for fragment coupling is HATU / DIEA, fragment I / fragment III: fragment II / fragment IV: HATU: DIEA = 1: 1.2: 1: 2.

[0024] Preferably, the solvent used for the cyclization of the crude linear carbetocin peptide in steps 1.4 and 2.4 is MeCN: H 2O=1: (0.5~3), DMSO, DMF or 6 M Gn∙HCl / 200 mM PBS, preferably MeCN: H 2 O=1:1; alkaline conditions are pH=8.0~9.0, and the reagents used to adjust pH are DIEA, NaOH or Na 2 CO 3 One of the above, preferably DIEA.

[0025] The beneficial effects of the present invention are as follows: it avoids the disadvantage of low purity of crude peptide synthesized by pure solid phase long fragments; it avoids the use of special cysteine ​​and expensive deprotection metal reagents; it improves the synthesis yield and purity, and is conducive to the large-scale industrial production of carbetocin. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The flow chart of the solid-liquid phase synthesis of carbetocin with two fragments via Asn ligation sites.

[0027] Figure 2 This is a flow chart of the solid-liquid phase synthesis of carbetocin via a Cys-linked site dual fragment.

[0028] Figure 3 It is a liquid chromatogram of carbetocin synthesized via the Asn attachment site.

[0029] Figure 4 It is a liquid chromatogram of carbetocin synthesized through the Cys attachment site.

[0030] Figure 5 This is the mass spectrum of cyclized carbetocin. DETAILED DESCRIPTION

[0031] The following is a detailed description of a method for synthesizing carbetocin in a double-fragment solid-liquid phase in conjunction with the embodiments of the present invention.

[0032] Example 1: Synthesis of polypeptide fragments Reference Figure 1, Fragment I: Weigh 2-Cl-CTC resin (1 mmol, 0.8 mmol / g) and place it in a peptide solid phase synthesizer, and add an appropriate amount of DMF / DCM (volume ratio of 1: 1) to swell. After the resin is completely swollen, the solvent is removed. The first amino acid is coupled to the resin using Fmoc-Asn(Trt)-OH (2 mmol, 2 equiv.) and DIEA (4 mmol, 4 equiv.). After 3 h of reaction, the resin is washed four times with DMF, and the unloaded resin is blocked with a DMF solution containing 5% MeOH and 10% DIEA by volume for 0.5 h. Other amino acids were coupled using Fmoc-AA-OH (2 mmol, 2 equiv.), DIC (2.2 mmol, 2.2 equiv.) and Oxyma (2 mmol, 2 equiv.), and the N-terminal amino acid was coupled using chlorobutyric acid (2 mmol, 2 equiv.), DIC (2.2 mmol, 2.2 equiv.) and HOBT (2 mmol, 2 equiv.). After solid phase synthesis, the obtained peptide resin was fully protected and cleaved in DCM containing 20% ​​HFIP by volume, filtered, and the solvent was concentrated to obtain the crude peptide fragment I, Cl-(CH 2 ) 3 CO-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-OH.

[0033] Fragment II: Weigh Sieber resin (1 mmol, 0.8 mmol / g) and place it in a peptide solid phase synthesizer, add an appropriate amount of DMF / DCM (volume ratio of 1: 1) to swell. After the resin is completely swollen, remove the solvent, add 20% piperidine by volume to remove the Fmoc protecting group, and react for 5 min (repeat twice). Then, the peptide sequence is synthesized using Fmoc-SPPS and coupled under the conditions of Fmoc-AA-OH (2 mmol, 2 equiv.), DIC (2.2 mmol, 2.2 equiv.) and Oxyma (2.0 mmol, 2 equiv.). After the solid phase synthesis, the obtained peptide resin was cleaved with DCM (20 mL) containing 5% TFA at 10°C for 10 min, and then cleaved with DCM (20 mL) containing 2% TFA at 10°C for 10 min and repeated twice. The cleavage solution was concentrated to obtain a crude peptide fragment II containing a Trt protective group on the side chain, NH 2 -Cys(Trt)-Pro-Leu-Gly-NH 2 .

[0034] Reference Figure 2 , Fragment III: Weigh 2-Cl-CTC resin (1 mmol, 0.8 mmol / g) and place it in a peptide solid phase synthesizer, and add an appropriate amount of DMF / DCM (volume ratio of 1: 1) to swell. After the resin is completely swollen, the solvent is removed, and the first amino acid is coupled to the resin using Fmoc-Cys(Trt)-OH (2 mmol, 2 equiv.) and DIEA (4 mmol, 4 equiv.). After 3 h of reaction, the resin is washed four times with DMF, and the unloaded resin is blocked with a DMF solution containing 5% MeOH and 10% DIEA by volume for 0.5 h. Other amino acids were coupled using Fmoc-AA-OH (2 mmol, 2 equiv.), DIC (2.2 mmol, 2.2 equiv.) and Oxyma (2 mmol, 2 equiv.), and the N-terminal amino acid was coupled using chlorobutyric acid (2 mmol, 2 equiv.), DIC (2.2 mmol, 2.2 equiv.) and HOBT (2 mmol, 2 equiv.). After solid phase synthesis, the obtained peptide resin was fully protected and cleaved in DCM containing 20% ​​HFIP by volume, filtered, and the solvent was concentrated to obtain the crude peptide fragment III, Cl-(CH 2 ) 3 CO-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-OH.

[0035] Fragment IV: Weigh Rink amide MBHA resin (1 mmol, 0.68 mmol / g) and place it in a peptide solid phase synthesizer, and add an appropriate amount of DMF / DCM (volume ratio of 1: 1) to swell. After the resin is completely swollen, the solvent is removed, and then 20% piperidine is added to remove the Fmoc protecting group and react for 5 min (repeat twice). Next, the peptide sequence is synthesized using Fmoc-SPPS and coupled under the conditions of Fmoc-AA-OH (2 mmol, 2 equiv.), DIC (2.2 mmol, 2.2 equiv.) and Oxyma (2 mmol, 2equiv.). After the solid phase synthesis is completed, the obtained peptide resin is quenched with TFA / TIS / H 2 O (volume ratio of 95:2.5: 2.5) lysis buffer (5 mL) was lysed at room temperature for 1 h, and the filtrate was concentrated to obtain crude peptide fragment IV, NH 2 -Pro-Leu-Gly-NH 2 .

[0036] Example 2: Reference Figure 1 or Figure 2 , the coupling agents for the condensation of amino acids (2.2 mmol, 2.2 equiv.) were HCTU (2 mmol, 2 equiv.) and DIEA (3.2 mmol, 3.2 equiv.), and the rest were the same as in Example 1.

[0037] Example 3: Reference Figure 1 or Figure 2 When the amino acid (2.2 mmol, 2.2 equiv.) was condensed, the coupling agents were PyBOP (2 mmol, 2 equiv.) and DIEA (3.2 mmol, 3.2 equiv.), and the rest was the same as in Example 1.

[0038] Example 4: Synthesis of Linear Carbetocin Crude Peptide HATU (0.38 g, 1 mmol) and DIEA (0.35 mL, 2 mmol) were added to a DMF (7.5 mL) solution containing crude peptide fragment I (1.14 g, 1 mmol). After the mixture was left at room temperature for activation for 5 min, a DMF (2 mL) solution containing crude peptide fragment II (0.83 g, 1.2 mmol) was added thereto, and the mixture was reacted at room temperature for 2 h. After the reaction, the reaction solution was added to ice brine to obtain a white solid, which was centrifuged, and the upper clear brine was poured off. Clean water was added again for washing, precipitation, and centrifugation (repeated 2 times). After the solid was dried, a crude carbetocin peptide with a side chain protection group was obtained. The crude carbetocin peptide was purified by lysing with 20 mL of TFA / TIS / H 2 O (95 / 2.5 / 2.5 volume), lysed for 1.5 h, filtered and dried to obtain the crude linear carbetocin peptide, Cl-(CH 2 ) 3 CO-Tyr(Me)-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH 2 .

[0039] Example 5: Synthesis of Linear Carbetocin Crude Peptide HATU (0.38 g, 1 mmol) and DIEA (0.35 mL, 2 mmol) were added to a DMF (8.75 mL) solution containing crude peptide fragment III (1.485 g, 1 mmol). After the mixture was left at room temperature for activation for 10 min, a DMF (2 mL) solution containing crude peptide fragment IV (0.34 mg, 1.2 mmol) was added thereto, and the mixture was reacted at room temperature for 2 h. After the reaction was completed, the reaction solution was added to ice brine to obtain a white solid, which was centrifuged, and the upper clear brine was poured off. Clean water was added again for washing, precipitation, and centrifugation (repeated 2 times). After the solid was dried, a crude carbetocin peptide with a side chain protection group was obtained. The crude carbetocin peptide was purified by lysing with 20 mL of TFA / TIS / H 2 O (95 / 2.5 / 2.5 volume), lysed for 1.5 h, filtered and dried to obtain the crude linear carbetocin peptide, Cl-(CH 2 ) 3 CO-Tyr(Me)-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH 2 .

[0040] Example 6: Reference Figure 1 or Figure 2 The coupling agents for synthesizing the crude linear carbetocin peptide are PyOxim (2 mmol, 2 equiv.) and DIEA (3.2 mmol, 3.2 equiv.), and the rest are the same as in Example 4 or Example 5.

[0041] Example 7: Reference Figure 1 or Figure 2 The coupling agents for synthesizing the crude linear carbetocin peptide are PyBOP (2 mmol, 2 equiv.) and DIEA (3.2 mmol, 3.2 equiv.), and the rest are the same as in Example 4 or Example 5.

[0042] Example 8: Intramolecular Cyclization Synthesis of Carbetocin The crude linear carbetocin peptide obtained in Example 4, Cl-(CH 2 ) 3 CO-Tyr(Me)-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH 2 Dissolved in acetonitrile / water (volume ratio 1: 1) at 10 mM, adjusted to pH 9.0 with DIEA, and reacted at room temperature for 72 hours. The reaction solution was purified by HPLC using a reverse C18 column to obtain 460 mg with a purity greater than 99%. The total yield of steps (3) and (4) was 46.6%. The final product was detected by HPLC-MS. The liquid chromatogram of the cyclized carbetocin is shown in Figure 3 The mass spectrum of cyclized carbetocin is shown in Figure 5 .

[0043] Example 9: Intramolecular Cyclization Synthesis of Carbetocin The crude linear carbetocin peptide obtained in Example 5, Cl-(CH 2 ) 3 CO-Tyr(Me)-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH 2 Dissolve in acetonitrile / water (volume ratio 1: 1) (10 mM), adjust pH to 9.0 with DIEA, and react at room temperature for 72 hours. The reaction solution was purified by HPLC using a reverse C18 column to obtain 439 mg with a purity greater than 99%. The total yield of steps (3) and (4) was 44.5%. The final product was detected by HPLC-MS. See the chromatogram for details. Figure 4 , mass spectrum see Figure 5 .

[0044] The following are the full names of the chemicals involved in the present invention: Abbreviations Representative meaning Fmoc Fmoc Trt Trityl Me methyl DIC -Diisopropylcarbodiimide HFIP Hexafluoroisopropanol TFA Trifluoroacetic acid TIS Triisopropylsilane DCC Dicyclohexylcarbodiimide DIC -Diisopropylcarbodiimide EDCI 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride HATU 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate HBTU -Benzotriazole-tetramethyluronium hexafluorophosphate TBTU 2-(1H-Benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate HCTU -(6-Chloro-1H-benzotriazol-1-yl)-tetramethyluronium hexafluorophosphate PyOxim Cyano(hydroxyimino)acetate ethyl-O2]tri-1-pyrrolidinyl hexafluorophosphate PyAop (7-Azabenzotriazole-1-oxy)tripyrrolylphosphonium hexafluorophosphate PyBop Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate HOAt 1-Hydroxy-7-azabenzotriazole HOB 1-Hydroxybenzotriazole Cl-HOBt 6-Chloro-1-hydroxybenzotriazole Oxyma Ethyl 2-oximecyanoacetate NMM N-Methylmorpholine DIEA Diisopropylethylamine DMAP 4-Dimethylaminopyridine MeCN Acetonitrile DCM Dichloromethane DMF N,N-Dimethylformamide The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for synthesizing carbetocin in a double-fragment solid-liquid phase, characterized in that: The synthesis of carbetocin is achieved by chemical coupling and cyclization of two fragments. The method couples two fragments at an Asn site or a Cys site, and then cyclizes to obtain carbetocin.

2. The method for synthesizing carbetocin by double-fragment solid-liquid phase according to claim 1, characterized in that: The method performs double-fragment coupling at the Asn site and then cyclizes to obtain carbetocin, specifically comprising the following steps: (1) Using 2-Cl-CTC resin as a carrier, the corresponding Fmoc-protected amino acids in the amino acid sequence of fragment I were sequentially connected through standard Fmoc solid-phase peptide synthesis, and then the chlorobutyric acid at the N-terminus was coupled using a condensation reagent. Finally, a DCM solution with a volume concentration of 20% HFIP or a DCM solution with a volume concentration of 1% TFA was used for cleavage to obtain a fragment I containing a side chain protection, Cl-(CH2)3CO-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-OH; (2) Using Sieber resin as a carrier, the corresponding Fmoc-protected amino acids in the amino acid sequence of fragment II were sequentially connected through standard Fmoc solid-phase peptide synthesis, and then cleaved using a DCM solution with a volume concentration of 2-5% TFA to obtain fragment II with a side chain protecting group and a free amino group at the N-terminus, NH2-Cys(Trt)-Pro-Leu-Gly-NH2; (3) After the fragments I and II are coupled in the liquid phase, the reaction solution is precipitated with saline, washed, and dried, and the obtained solid is cleaved with a cleavage solution, precipitated with cold ether, filtered, and dried to obtain a crude linear oxytocin peptide; (4) Cyclizing the obtained linear carbetocin crude peptide under alkaline conditions in a solvent to obtain carbetocin crude peptide, purifying, and freeze-drying to obtain carbetocin.

3. The method for synthesizing carbetocin by double-fragment solid-liquid phase according to claim 1, characterized in that: The double-fragment coupling is performed at the Cys site, followed by cyclization to obtain carbetocin, which specifically comprises the following steps: (1) Using 2-Cl-CTC resin as a carrier, the corresponding Fmoc-protected amino acids in the amino acid sequence of fragment III were sequentially connected through standard Fmoc solid-phase peptide synthesis, and then the chlorobutyric acid at the N-terminus was coupled using a condensation reagent. Finally, a DCM solution with a volume concentration of 20% HFIP or a DCM solution with a volume concentration of 1% TFA was used for cleavage to obtain a fragment III containing a side chain protection, Cl-(CH2)3CO-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-OH; (2) Using Rink Amide resin as a carrier, the corresponding Fmoc-protected amino acids in the amino acid sequence of fragment IV were sequentially connected through standard Fmoc solid-phase peptide synthesis, and fragment IV with a side chain protecting group and a free amino group at the N-terminus was obtained after cleavage using a cleavage solution, NH2-Pro-Leu-Gly-NH2; (3) After the fragment III and the fragment IV are coupled in the liquid phase, the reaction solution is precipitated with saline, washed, and dried. The obtained solid is cleaved with a cleavage solution, precipitated with cold ether, filtered, and dried to obtain a crude linear oxytocin peptide; (4) Cyclizing the obtained linear carbetocin crude peptide under alkaline conditions in a solvent to obtain carbetocin crude peptide, purifying, and freeze-drying to obtain carbetocin.

4. The method for synthesizing carbetocin by double-fragment solid-liquid phase according to claim 2, characterized in that: The lysis solution used in step (3) is TFA, with a volume concentration of 90-95%.

5. The method for synthesizing carbetocin by double-fragment solid-liquid phase according to claim 3, characterized in that: The lysis solution used in steps (2) and (3) is TFA, with a volume concentration of 90-95%.

6. A method for synthesizing carbetocin by double-fragment solid-liquid phase according to claim 2 or 3, characterized in that: The condensation reagent used in the synthesis of the fragments in steps (1) and (2) and the coupling of the fragments in step (3) is one of DIC, DCC, EDCI, HATU, TBTU, HCTU, HBTU, PyOxim, PyAop or PyBop. The auxiliary agent used in the condensation is one of HOAt, HOBt, Cl-HOBt, DIEA, Oxyma or NMM. The solvent is DMF. The amount of each component used in the coupling is calculated by molar ratio, amino acid / chlorobutyric acid: condensation reagent: auxiliary agent = 1: 1: (1-2), fragment I / fragment III: fragment II / fragment IV: condensation reagent: auxiliary agent = 1: 1: 1: (1-2).

7. The method for synthesizing carbetocin by double-fragment solid-liquid phase according to claim 6, characterized in that: The condensation reagent / auxiliary agent for amino acid condensation is DIC / Oxyma, and the amount of each component used during coupling is calculated by molar ratio, amino acid: DIC: Oxyma = 1: 1.2: 1; the condensation reagent / auxiliary agent for chlorobutyric acid condensation is DIC / HOBt, chlorobutyric acid: DIC: HOBt = 1:1.2: 1; the condensation reagent / auxiliary agent for fragment coupling is HATU / DIEA, fragment I / fragment III: fragment II / fragment IV: HATU: DIEA = 1: 1.2: 1:

2.

8. A method for synthesizing carbetocin by double-fragment solid-liquid phase according to claim 2 or 3, characterized in that: The dosage is based on a molar ratio. The solvent used for the cyclization of the crude linear carbetocin peptide in step (4) is one of MeCN: H2O=1: (0.5~3), DMSO, DMF or 6 M Gn∙HCl / 200 mM PBS. The alkaline condition is pH=8.0~9.0, and the reagent used to adjust the pH is one of DIEA, NaOH or Na2CO3.

9. The method for synthesizing carbetocin by double-fragment solid-liquid phase according to claim 8, characterized in that: The dosage is based on molar ratio. The solvent used for the cyclization of the crude linear carbetocin peptide is MeCN: H 2O=1:1, and the reagent used to adjust pH is DIEA.

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