A method for the directed synthesis of a polypeptide dimer

Abstract

The present invention relates to the field of polypeptide synthesis, and particularly to a method for the directional synthesis of polypeptide dimers. The method comprises the following steps: 1) preparing linear peptide resins A containing aa1(Trt) and aa2(Acm), and linear peptide resins B containing aa1(Acm) and aa2(Trt); 2) cleaving resins A and B with cleavage reagent 1 to obtain linear peptides A1 containing aa1(activated group) and aa2(Acm), and linear peptides B1 containing aa1(Acm) and aa2(activated group); cleaving resins A and B with cleavage reagent 2 to obtain linear peptides A2 containing aa1 and aa2(Acm), and linear peptides B2 containing aa1(Acm) and aa2; 3) cyclizing the linear peptides containing the activated group and the linear peptides without the activated group for the first time; 4) removing Acm and cyclizing for the second time to obtain cis or trans polypeptide dimers. By double selection of the Cys side chain protecting group of the linear peptide resin and the cleavage path, the present invention realizes the directional formation of intermolecular disulfide bonds by means of stepwise oxidation. The obtained main product is single, with high purity and high yield, which is beneficial to the large-scale industrial production of polypeptide dimers.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide synthesis, and particularly relates to a method for the directed synthesis of polypeptide dimers. Background Art

[0002] In polypeptide synthesis, two thiol side chains can be oxidized into corresponding disulfide bonds by an appropriate oxidant. The study of methods for disulfide bond formation is one of the hotspots and difficulties in the field of polypeptide synthesis. The air oxidation method, potassium ferricyanide oxidation method, iodine oxidation method, hydrogen peroxide oxidation method, DMSO oxidation method, thallium trifluoroacetate oxidation method, and Npys oxidation method are commonly used methods in polypeptide synthesis applications. Some polypeptides often need to form one or more pairs of disulfide bonds, and disulfide bonds can be formed intramolecularly or intermolecularly, which can be achieved by one-step oxidation method and stepwise oxidation method. If more than two pairs of disulfide bonds need to be selectively formed between thiols, the one-step oxidation method requires that the structure has a thermodynamically stable natural conformation, otherwise it is difficult to ensure the formation of correctly paired disulfide bonds. The stepwise oxidation method uses more than two different thiol protecting groups and takes advantage of their property differences to stepwise oxidize to form more than two pairs of disulfide bonds. Cyclic polypeptides containing a pair of disulfide bonds, such as oxytocin, terlipressin, octreotide, atosiban, etc., may polymerize to form dimers during synthesis and storage, which become important impurities affecting the quality of drugs. Impurity research is an important part of drug quality research and one of the key factors for drug quality assurance. In order to ensure the safety of clinical medication, it is necessary to conduct a detailed analysis of process impurities generated during synthesis and degradation impurities generated during storage. Such dimer impurities have α configuration and β configuration, that is, cis-dimer and trans-dimer. In order to facilitate the research of impurities, the directed synthesis of dimer impurities is particularly crucial.

[0003] Patent application document CN110218242 synthesizes linear peptide A: Mpa(Acm)-D-Tyr(Et)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH2 or linear peptide B: Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys(Acm)-Pro-Orn-Gly-NH2; performs liquid-phase cyclization on linear peptide A or linear peptide B with hydrogen peroxide, and then performs secondary cyclization with an iodine / methanol solution to obtain atosiban cis-dimer (α-Dimer), and its structural formula is: By synthesizing linear peptide A: Mpa(Acm)-D-Tyr(Et)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH2 and linear peptide B: Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys(Acm)-Pro-Orn-Gly-NH2; mixing linear peptide A and linear peptide B (molar ratio 1:1), performing liquid-phase cyclization with hydrogen peroxide, and then performing secondary cyclization with an iodine / methanol solution, atosiban trans-dimer (β-Dimer) is obtained, and its structural formula is: However, when synthesizing the cis-dimer by this method, due to the strong oxidizing property of hydrogen peroxide, more small impurities are easily generated, and there is also a risk of over-oxidation, resulting in a low yield; when synthesizing the trans-dimer by this method, it is found that reactions occur between linear peptide A and linear peptide A or between linear peptide B and linear peptide B, so cis-dimer is still produced and directional synthesis cannot be achieved. Summary of the Invention

[0004] Aiming at the problems of many impurities, low yield, and inability to achieve directional synthesis existing in the synthesis process of the existing polypeptide dimers, the present invention provides a method for the directional synthesis of polypeptide dimers. The reaction conditions of this method are mild, the reaction time is short, and the obtained product has high purity and can be used as an impurity reference substance for the quality research of polypeptide bulk drugs and preparations, thus effectively ensuring and controlling the quality of polypeptide drugs.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] A method for the directional synthesis of polypeptide dimers, comprising the following steps: (1) preparing linear peptide resin A containing aa1(Trt) and aa2(Acm), and linear peptide resin B containing aa1(Acm) and aa2(Trt); (2) using cleavage reagent 1 to cleave resin A and B to obtain linear peptide A1 containing aa1(activating group) and aa2(Acm), and linear peptide B1 containing aa1(Acm) and aa2(activating group); using cleavage reagent 2 to cleave resin A and B to obtain linear peptide A2 containing aa1 and aa2(Acm), and linear peptide B2 containing aa1(Acm) and aa2; (3) performing the first cyclization of the linear peptide containing the activating group and the linear peptide without the activating group in an acidic solution; (4) removing Acm and performing the second cyclization to obtain cis- or trans-polypeptide dimer. In some embodiments of the present invention, aa1 and aa2 are selected from Cys and Mpa.

[0007] In some embodiments of the present invention, the polypeptide is selected from polypeptides containing two sulfhydryl groups on the peptide chain, such as oxytocin, atosiban, octreotide, terlipressin, desmopressin, ulinastatin, somatostatin, etc.

[0008] Preferably, the cleavage reagent 1 is selected from a mixed solution of TFA, pyridyl disulfide compounds, and T; the cleavage reagent 2 is selected from a mixed solution of TFA and T; where T is selected from one or more compounds among Tis, Mpr, m-cresol, Phenol, and H2O.

[0009] As a preferred embodiment, the disulfide compounds are selected from 2,2'-dithiobis(5-nitropyridine), dithiodipyridine, 2,2'-dithiopyridine, and 4,4'-dithiopyridine. Further preferably, 2,2′-dithiopyridine is selected. 2,2′-dithiopyridine has a relatively high selectivity in the reaction with thiol groups and the fastest rate in the first cyclization reaction.

[0010] As a preferred embodiment, the acidic solution is selected from acetic acid, hydrochloric acid, phosphoric acid, or their buffer salt solutions. Further preferably, acetic acid is selected. Linear peptides have good solubility in aqueous acetic acid solution, and the aqueous acetic acid solution can continue to be used as the reaction solvent for the second cyclization using the iodine oxidation method.

[0011] In some embodiments of the present invention, the second cyclization is carried out by the iodine oxidation method or the hydrogen peroxide oxidation method.

[0012] In some embodiments of the present invention, the disulfide compound is preferably 2,2'-dithiopyridine. The synthesis of the polypeptide dimer is specifically as follows: (1) Prepare linear peptide resin A containing aa1(Trt) and aa2(Acm), and linear peptide resin B containing aa1(Acm) and aa2(Trt); (2) Use cleavage reagent 1 to cleave resin A and B to obtain linear peptide A1 containing aa1(S-Pyr) and aa2(Acm), and linear peptide B1 containing aa1(Acm) and aa2(S-Pyr); Use cleavage reagent 2 to cleave resin A and B to obtain linear peptide A2 containing aa1 and aa2(Acm), and linear peptide B2 containing aa1(Acm) and aa2; (3) First cyclize the linear peptide containing S-Pyr and the linear peptide without S-Pyr in an acidic solution; (4) Remove Acm and perform the second cyclization to obtain cis or trans polypeptide dimer.

[0013] Furthermore, the synthesis of the cis polypeptide dimer is specifically as follows: (1) Prepare linear peptide resin A containing aa1(Trt) and aa2(Acm); (2) Use cleavage reagent 1 to cleave resin A to obtain linear peptide A1 containing aa1(S-Pyr) and aa2(Acm); Use cleavage reagent 2 to cleave resin A to obtain linear peptide A2 containing aa1 and aa2(Acm); (3) Take linear peptides A1 and A2 for the first cyclization in an acidic solution; (4) Remove Acm and perform the second cyclization to obtain the cis polypeptide dimer;

[0014] Or (1) prepare a linear peptide resin B containing aa1 (Acm) and aa2 (Trt); (2) cleave resin B with cleavage reagent 1 to obtain a linear peptide B1 containing aa1 (Acm) and aa2 (S-Pyr); cleave resin B with cleavage reagent 2 to obtain a linear peptide B2 containing aa1 (Acm) and aa2; (3) take linear peptides B1 and B2 and cyclize them for the first time in an acidic solution; (4) remove Acm and cyclize them for the second time to obtain a cis-polypeptide dimer.

[0015] Furthermore, the synthesis of the trans-polypeptide dimer is specifically carried out in the following steps: (1) prepare a linear peptide resin A containing aa1 (Trt) and aa2 (Acm), and a linear peptide resin B containing aa1 (Acm) and aa2 (Trt); (2) cleave resins A and B with cleavage reagent 1 to obtain a linear peptide A1 containing aa1 (S-Pyr) and aa2 (Acm), and a linear peptide B1 containing aa1 (Acm) and aa2 (S-Pyr); cleave resins A and B with cleavage reagent 2 to obtain a linear peptide A2 containing aa1 and aa2 (Acm), and a linear peptide B2 containing aa1 (Acm) and aa2; (3) take linear peptides A1 and B2 and cyclize them for the first time in an acidic solution; (4) remove Acm and cyclize them for the second time to obtain a trans-polypeptide dimer;

[0016] Or (3) take linear peptides A2 and B1 and cyclize them for the first time in an acidic solution; (4) remove Acm and cyclize them for the second time to obtain a trans-polypeptide dimer.

[0017] The invention realizes the directional formation of intermolecular disulfide bonds by means of stepwise oxidation through the dual selection of the Cys side-chain protecting group of the linear peptide resin and the cleavage path. In the technical solution of the present invention, the formation of the first pair of disulfide bonds adopts a pyridyl disulfide compound to activate the mercapto side chain of one linear peptide and then carry out a directional reaction with another linear peptide with an exposed mercapto group, effectively avoiding the misconnection of intramolecular or intermolecular disulfide bonds during the synthesis of the dimer. The obtained main product is single, with high purity and high yield, and the reaction conditions are mild and the reaction time is short, which is conducive to the kilogram-scale and industrial production of polypeptide dimers. Specific Embodiments

[0018] The technical solution of the present invention will be described below in conjunction with specific embodiments. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

[0019] Example 1. Synthesis of Octreotide Cis-Dimer

[0020] Synthesis of Octreotide Linear Peptide Resin A

[0021] Weigh 32.2 g of Amide AM resin (20 mmol, substitution value 0.65 mmol / g), add it to a peptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and then drain. Wash it twice with DMF and then drain. Weigh fluorenylmethyloxycarbonyl thiaminol p-carboxybenzene acetal (27.59 g, 3.0 eq.) and HOBt (8.91 g, 3.3 eq.), dissolve them in 150 ml of DMF, add DIC (14.2 ml, 4.5 eq.) at 0 - 10 °C to activate for 4 min, add it to the synthesis reaction column, and react for 2 - 4 h under nitrogen protection at 25 ± 5 °C. Drain, wash 4 times with DMF. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF.

[0022] Repeat the above steps to couple Fmoc-Cys(Acm)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Cys(Trt)-OH, and Fmoc-D-Phe-OH in sequence. Remove the Fmoc protecting group, then wash 4 times with DMF, 3 times with DCM, and 3 times with MeOH. After drying, 60.08 g of octreotide linear peptide resin A is obtained: D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-ol-Amide AM resin.

[0023] Synthesis of octreotide linear peptide A1

[0024] Weigh 30.04 g of the above-mentioned octreotide linear peptide resin A, add it to 300.0 ml of cleavage reagent 1 [(TFA / Tis / H2O / m-cresol = 94.0 / 2.5 / 2.5 / 1.0), 2,2'-dithiobis(5-nitropyridine) (6.20 g, 2 eq.)], and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter, and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, 17.28 g of octreotide linear peptide A1 is obtained:

[0025]

[0026] Synthesis of octreotide linear peptide A2

[0027] Weigh 30.04 g of the above-mentioned octreotide linear peptide resin A, add it to 300.0 ml of cleavage reagent 2 (TFA / Tis / H2O / m-cresol = 94.0 / 2.5 / 2.5 / 1.0), and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter, and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of ice-cold isopropyl ether for precipitation, precipitate the solid, centrifuge, and wash to obtain 17.02 g of octreotide linear peptide A2: D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys(Acm)-Thr-OH.

[0028] The first cyclization

[0029] Weigh 17.28 g of the above-mentioned octreotide linear peptide A1, add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% aqueous acetic acid solution;

[0030] Weigh 17.02 g of octreotide linear peptide A2, add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% aqueous acetic acid solution;

[0031] Add the above-mentioned aqueous acetic acid solution of linear peptide A1 to the aqueous acetic acid solution of linear peptide A2, stir and react at room temperature for 1 - 2 h to form intermediate C. After the reaction is completed, filter the crude intermediate C solution through a 0.45 μm organic membrane, and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% aqueous TFA solution and acetonitrile for gradient elution, combine the fractions with a purity > 80% to obtain the octreotide dimer impurity intermediate C:

[0032]

[0033] The second cyclization

[0034] Add 400 ml of acetic acid to the purified octreotide dimer impurity intermediate C solution, and stir evenly;

[0035] Slowly add an iodine ethanol solution, keep the color of the solution light yellow and not fade, stir and react at room temperature for about 16 - 20 h. After the reaction is completed, add ascorbic acid (Vc) to quench the reaction to obtain a crude solution of octreotide cis-dimer (α-Dimer). The HPLC purity of the crude product is 86.42%, the front impurity is 0.47%, and the back impurity is 1.74%.

[0036] Filter the crude solution of octreotide cis-dimer (α-Dimer) through a 0.45 μm organic membrane, and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Gradient elution was carried out using 0.1% acetic acid aqueous solution and acetonitrile. The fractions with purity >90% were combined to obtain the dimer impurity sample. The sample was concentrated by rotary evaporation in a 32°C water bath to remove the organic solvent, and after freeze-drying, octreotide cis-dimer (α-Dimer) was obtained:

[0037]

[0038] The weight of octreotide cis-dimer (α-Dimer) was weighed as 13.09 g, the total yield was 64.23%, the HPLC purity was 97.82%, and the maximum single impurity was 0.48%.

[0039] Example 2. Synthesis of octreotide trans-dimer

[0040] Synthesis of octreotide linear peptide resin A

[0041] Weigh 16.1 g of Amide AM resin (10 mmol, substitution value 0.65 mmol / g), add it to the polypeptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and drain. Wash twice with DMF and drain. Weigh 9-fluorenylmethoxycarbonylthreoninol p-carboxybenzene acetal (13.80 g, 3.0 eq.), HOBt (4.45 g, 3.3 eq.) and dissolve them in 150 ml of DMF. Add DIC (7.1 ml, 4.5 eq.) at 0-10°C to activate for 4 min, add it to the synthesis reaction column, and react for 2-4 h under nitrogen protection at 25±5°C. Drain and wash 4 times with DMF. Add 20% PIPE / DMF to remove the Fmoc protection twice, and the deprotection times are 5 min + 10 min respectively, and wash 6 times with DMF.

[0042] Repeat the above steps to couple Fmoc-Cys(Acm)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Cys(Trt)-OH and Fmoc-D-Phe-OH in turn. Remove the Fmoc protecting group, and then wash 4 times with DMF, 3 times with DCM, and 3 times with MeOH. After drying, 30.44 g of octreotide linear peptide resin A: D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-ol–Amide AM resin was obtained.

[0043] Synthesis of octreotide linear peptide resin B

[0044] Weigh 16.1 g of Amide AM resin (20 mmol, substitution value 0.65 mmol / g), add it to a peptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and drain. Wash twice with DMF and drain. Weigh fluorenylmethyloxycarbonylthreoninol p-carboxybenzene acetal (13.80 g, 3.0 eq.), HOBt (4.45 g, 3.3 eq.) and dissolve them in 150 ml of DMF. Add DIC (7.1 ml, 4.5 eq.) at 0 - 10 °C to activate for 4 min, then add it to the synthesis reaction column and react for 2 - 4 h under nitrogen protection at 25 ± 5 °C. Drain, wash 4 times with DMF. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF.

[0045] Repeat the above steps to couple Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Cys(Acm)-OH and Fmoc-D-Phe-OH in sequence. Remove the Fmoc protecting group, then wash 4 times with DMF, 3 times with DCM, 3 times with MeOH, and after drying, obtain 29.96 g of octreotide linear peptide resin B: D-Phe-Cys(Acm)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr-ol-Amide AM resin.

[0046] Synthesis of octreotide linear peptide A1

[0047] Weigh 30.44 g of the above octreotide linear peptide resin A, add it to 300.0 ml of cleavage reagent 1 [(TFA / Tis / H2O / m-cresol = 94.0 / 2.5 / 2.5 / 1.0), 2,2'-dithiobis(5-nitropyridine) (6.20 g, 2 eq.)], and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of ice-cold isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, obtain 17.28 g of octreotide linear peptide A1:

[0048]

[0049] Synthesis of octreotide linear peptide B2

[0050] Weigh 29.96 g of the above-mentioned octreotide linear peptide resin B, add it to 300.0 ml of cleavage reagent 2 (TFA / Tis / H2O / m-cresol = 94.0 / 2.5 / 2.5 / 1.0), and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of ice-cold isopropyl ether for precipitation, precipitate the solid, centrifuge, and wash to obtain 17.02 g of octreotide linear peptide B2: D-Phe-Cys(Acm)-Phe-D-Trp-Lys-Thr-Cys-Thr-OH.

[0051] First cyclization

[0052] Weigh 17.28 g of the above-mentioned octreotide linear peptide A1, add 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution;

[0053] Weigh 17.02 g of octreotide linear peptide B2, add 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution;

[0054] Add the above acetic acid aqueous solution of linear peptide A1 to the acetic acid aqueous solution of linear peptide B2, stir and react at room temperature for 1 - 2 h to form intermediate D. After the reaction is completed, filter the crude intermediate D solution through a 0.45 μm organic membrane, and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% TFA aqueous solution and acetonitrile for gradient elution, combine the fractions with a purity > 80% to obtain the octreotide dimer impurity intermediate D.

[0055]

[0056] Second cyclization

[0057] Add 400 ml of acetic acid to the purified intermediate D solution and stir evenly;

[0058] Slowly add an iodine ethanol solution, keep the color of the solution light yellow and not fade, stir and react at room temperature for about 16 - 20 h. After the reaction is completed, add ascorbic acid (Vc) to quench the reaction to obtain a crude solution of octreotide trans-dimer (β-Dimer). The HPLC purity of the crude product is 78.39%, the front impurity is 0.35%, and the back impurity is 2.03%.

[0059] Filter the crude solution of octreotide trans-dimer (β-Dimer) through a 0.45 μm organic membrane, and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Gradient elution was carried out using 0.1% aqueous acetic acid solution and acetonitrile. The fractions with a purity > 90% were combined to obtain the dimer impurity sample. The sample was concentrated by rotary evaporation in a 32°C water bath to remove the organic solvent, and after freeze-drying, octreotide trans-dimer (β-Dimer) was obtained:

[0060]

[0061] The weight of octreotide trans-dimer (β-Dimer) was weighed as 11.74 g, the total yield was 57.58%, the HPLC purity was 98.84%, and the maximum single impurity was 0.86%.

[0062] Example 3: Synthesis of oxytocin cis-dimer

[0063] Synthesis of oxytocin linear peptide resin A

[0064] Weigh 25.6 g of Rink Amide MBHA resin (20 mmol, substitution value 0.78 mmol / g), add it to the polypeptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and drain. Wash with DMF twice and drain. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash with DMF 6 times. Weigh Fmoc-Gly-OH (11.90 g, 2.0 eq.), HOBt (5.94 g, 2.2 eq.) and dissolve them in 100 ml of DMF. Add DIC (9.5 ml, 3.0 eq.) at 0 - 10°C to activate for 4 min, add it to the synthesis reaction column, and react under nitrogen protection at 25 ± 5°C for 2 - 4 h. Drain and wash with DMF 4 times.

[0065] Repeat the above steps to couple Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH and Fmoc-Cys(Trt)-OH in sequence. Remove the Fmoc protecting group, and then wash with DMF 4 times, DCM 3 times, and MeOH 3 times. After drying, 62.52 g of oxytocin linear peptide resin A was obtained: H-Cys(Trt)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-Leu-Gly-Rink Amide MBHA resin.

[0066] Synthesis of oxytocin linear peptide A1

[0067] Weigh 31.26 g of the above-mentioned oxytocin linear peptide resin A containing aa1(Trt) and aa2(Acm), and add it to 312.6 ml of cleavage reagent 1 [(TFA / Tis / H2O = 95.0 / 2.5 / 2.5), 2,2-dithiopyridine (4.41 g, 2 eq.)]. Carry out the cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, obtain 18.38 g of oxytocin linear peptide A1: H-Cys(S-Pyr)-Tyr-Ile-Gln-Asn-Cys(Acm)-Pro-Leu-Gly-NH2, that is

[0068]

[0069] Synthesis of Oxytocin Linear Peptide A2

[0070] Weigh 31.26 g of the above-mentioned oxytocin linear peptide resin A, and add it to 312.6 ml of cleavage reagent 2 (TFA / Tis / H2O = 95.0 / 2.5 / 2.5). Carry out the cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, obtain 18.04 g of oxytocin linear peptide A2: H-Cys-Tyr-Ile-Gln-Asn-Cys(Acm)-Pro-Leu-Gly-NH2.

[0071] First Cyclization

[0072] Weigh 18.38 g of the above-mentioned oxytocin linear peptide A1, and add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution;

[0073] Weigh 18.04 g of oxytocin linear peptide A2, and add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution;

[0074] Add the above-mentioned acetic acid aqueous solution of linear peptide A1 to the acetic acid aqueous solution of linear peptide A2, and stir at room temperature for 1 - 2 h to form intermediate E. After the reaction is completed, filter the crude intermediate E solution through a 0.45 μm organic membrane, and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% TFA aqueous solution and acetonitrile for gradient elution, and combine the fractions with a purity > 80% to obtain the oxytocin dimer impurity intermediate E.

[0075]

[0076] Second Cyclization

[0077] Add the purified intermediate E solution, and then add 400 ml of acetic acid and stir evenly.

[0078] Slowly add the iodine ethanol solution, keep the color of the solution light yellow and not fading, stir and react at room temperature for about 16 - 20 h. After the reaction is completed, add ascorbic acid (Vc) to quench the reaction to obtain a crude solution of oxytocin cis-dimer (α-Dimer). The HPLC purity of the crude product is 84.84%, the front impurity is 1.72%, and the back impurity is 0.50%.

[0079] The crude solution of oxytocin cis-dimer (α-Dimer) is filtered through a 0.45 μm organic membrane and purified and separated by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% aqueous acetic acid solution and acetonitrile for gradient elution. Combine the fractions with a purity > 90% to obtain a dimer impurity combined sample. The combined sample is rotary evaporated and concentrated at 32 °C in a water bath to remove the organic solvent, and then freeze-dried to obtain oxytocin cis-dimer (α-Dimer):

[0080]

[0081] Weigh the weight of oxytocin cis-dimer (α-Dimer) as 13.79 g, the total yield is 68.47%, the HPLC purity is 98.73%, and the maximum single impurity is 0.42%.

[0082] Example 4. Synthesis of oxytocin trans-dimer

[0083] Synthesis of oxytocin linear peptide resin A

[0084] Weigh 12.8 g of Rink Amide MBHA resin (10 mmol, substitution value 0.78 mmol / g), add it to the polypeptide synthesis reaction column, add 100 ml of DMF to swell for 30 min, and drain. Wash twice with DMF and drain. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF. Weigh Fmoc-Gly-OH (5.95 g, 2.0 eq.), HOBt (2.97 g, 2.2 eq.) and dissolve them in 50 ml of DMF. Add DIC (4.7 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, add it to the synthesis reaction column, and react under nitrogen protection at 25 ± 5 °C for 2 - 4 h. Drain and wash 4 times with DMF.

[0085] Repeat the above steps to couple Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Trt)-OH in sequence. Remove the Fmoc protecting group, and then wash with DMF 4 times, DCM 3 times, and MeOH 3 times. After drying, 32.04 g of oxytocin linear peptide resin A: H-Cys(Trt)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-Leu-Gly-Rink Amide MBHA resin is obtained.

[0086] Synthesis of oxytocin linear peptide resin B

[0087] Weigh 12.8 g of Rink Amide MBHA resin (10 mmol, substitution value 0.78 mmol / g), add it to the polypeptide synthesis reaction column, add 100 ml of DMF to swell for 30 min, and drain. Wash with DMF twice and drain. Weigh Fmoc-Gly-OH (5.95 g, 2.0 eq.) and HOBt (2.97 g, 2.2 eq.), dissolve them in 50 ml of DMF, add DIC (4.7 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, add it to the synthesis reaction column, and react for 2 - 4 h under nitrogen protection at 25 ± 5 °C. Drain, and wash with DMF 4 times. Add 20% PIPE / DMF to remove the Fmoc protecting group twice, with the deprotection times being 5 min + 10 min respectively, and wash with DMF 6 times.

[0088] Repeat the above steps to couple Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Acm)-OH in sequence. Remove the Fmoc protecting group, and then wash with DMF 4 times, DCM 3 times, and MeOH 3 times. After drying, 31.78 g of oxytocin peptide resin B: H-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-RinkAmide MBHA resin is obtained.

[0089] Synthesis of oxytocin linear peptide A1

[0090] Weigh 32.04 g of the oxytocin linear peptide resin A containing aa1(Trt) and aa2(Acm) as described above, and add it to 320.4 ml of cleavage reagent 1 [(TFA / Tis / H2O = 95.0 / 2.5 / 2.5), 2,2-dithiopyridine (4.41 g, 2 eq.)]. Carry out the cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of ice-cold isopropyl ether for precipitation. The precipitated solid is centrifuged, washed, and 18.62 g of oxytocin linear peptide A1: H-Cys(S-Pyr)-Tyr-Ile-Gln-Asn-Cys(Acm)-Pro-Leu-Gly-NH2 is obtained, that is

[0091]

[0092] Synthesis of Oxytocin Linear Peptide B2

[0093] Weigh 32.78 g of the oxytocin linear peptide resin B as described above, and add it to 327.8 ml of cleavage reagent 2 (TFA / Tis / H2O = 95.0 / 2.5 / 2.5). Carry out the cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of ice-cold isopropyl ether for precipitation. The precipitated solid is centrifuged, washed, and 17.86 g of oxytocin linear peptide B2: H-Cys(Acm)-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 is obtained.

[0094] First Cyclization

[0095] Weigh 18.62 g of the oxytocin linear peptide A1 as described above, and add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% aqueous acetic acid solution;

[0096] Weigh 17.86 g of the oxytocin linear peptide B2, and add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% aqueous acetic acid solution;

[0097] Add the above-mentioned aqueous acetic acid solution of linear peptide A1 to the aqueous acetic acid solution of linear peptide B2, stir and react at room temperature for 1 - 2 h to form intermediate F. After the reaction is completed, the crude intermediate F solution is filtered through a 0.45 μm organic membrane and purified and separated by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% aqueous TFA solution and acetonitrile for gradient elution, and combine the fractions with a purity > 80% to obtain the oxytocin dimer impurity intermediate F.

[0098]

[0099] Second Cyclization

[0100] Add the purified intermediate F solution, and then add 400 ml of acetic acid and stir evenly.

[0101] Slowly add the iodine ethanol solution, keep the color of the solution light yellow and not fading, stir and react at room temperature for about 16 - 20 h. After the reaction is completed, add ascorbic acid (Vc) to quench the reaction to obtain a crude solution of oxytocin trans - dimer (β - Dimer). The HPLC purity of the crude product is 70.28%, the front impurity is 1.89%, and the rear impurity is 0.86%.

[0102] The crude solution of oxytocin trans - dimer (β - Dimer) is filtered through a 0.45 μm organic membrane and purified and separated by reverse - phase liquid chromatography. The packing material is reverse - phase C18, with a particle size of 8 μm and a pore size of Use an aqueous solution of 0.1% acetic acid and acetonitrile for gradient elution, combine the fractions with a purity > 90% to obtain a dimer impurity sample. The sample is rotary evaporated and concentrated in a 32 °C water bath to remove the organic solvent, and then freeze - dried to obtain oxytocin trans - dimer (β - Dimer):

[0103]

[0104] Weigh the weight of oxytocin trans - dimer (β - Dimer) as 12.94 g, the total yield is 64.23%, the HPLC purity is 98.63%, and the maximum single impurity is 0.37%.

[0105] Example 5: Synthesis of atosiban cis - dimer

[0106] Synthesis of atosiban linear peptide resin B

[0107] Weigh 21.0 g of Rink Amide AM resin (20 mmol, substitution value 0.95 mmol / g), add it to the polypeptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and drain. Then wash twice with DMF and drain. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF. Weigh Fmoc - Gly - OH (11.90 g, 2.0 eq.), HOBt (5.94 g, 2.2 eq.) and dissolve them in 100 ml of DMF, add DIC (9.5 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, add it to the synthesis reaction column, and react under nitrogen protection at 25 ± 5 °C for 2 - 4 h. Drain and wash 4 times with DMF.

[0108] Repeat the above steps to couple Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, and Mpa(Acm)-OH in sequence. Remove the Fmoc protecting group, and then wash with DMF 4 times, DCM 3 times, and MeOH 3 times. After drying, 41.38 g of atosiban linear peptide resin B: Mpa(Acm)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-Rink Amide AM resin is obtained. Synthesis of atosiban linear peptide B1

[0109] Weigh 20.69 g of the above atosiban linear peptide resin B and add it to 206.9 ml of cleavage reagent 1 [(TFA / Tis / H2O / m-cresol = 93.5 / 3.0 / 2.5 / 2.0), 2,2-dithiopyridine (4.41 g, 2 eq.)]. Carry out the cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 2500 ml of frozen isopropyl ether for precipitation. The solid is precipitated, centrifuged, and washed to obtain 10.13 g of atosiban linear peptide B1: Mpa(Acm)-D-Tyr(Et)-Ile-Thr-Asn-Cys(S-Pyr)-Pro-Orn-Gly-NH2, that is

[0110]

[0111] Synthesis of atosiban linear peptide B2

[0112] Weigh 20.69 g of the above atosiban linear peptide resin B and add it to 206.9 ml of cleavage reagent 2 (TFA / Tis / H2O / m-cresol = 93.5 / 3.0 / 2.5 / 2.0). Carry out the cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of frozen isopropyl ether for precipitation. The solid is precipitated, centrifuged, and washed to obtain 9.96 g of oxytocin linear peptide B2: Mpa(Acm)-D-Tyr(Et)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH2.

[0113] First cyclization

[0114] Weigh 10.13 g of the above atosiban linear peptide B1 and add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution;

[0115] Weigh 9.96 g of atosiban linear peptide B2, and add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution;

[0116] Add the above-mentioned acetic acid aqueous solution of linear peptide B1 to the acetic acid aqueous solution of linear peptide B2, and stir and react at room temperature for 1 - 2 h to form intermediate G. After the reaction is completed, the crude solution of intermediate G is filtered through a 0.45 μm organic membrane, and purified and separated by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% aqueous TFA solution and acetonitrile for gradient elution, combine the fractions with a purity > 80% to obtain the atosiban dimer impurity intermediate G.

[0117]

[0118] Second cyclization

[0119] Add 25% ammonia water dropwise to the purified intermediate G solution to adjust the pH = 8.0 ± 0.5;

[0120] Slowly add 1.2 ml of 30% hydrogen peroxide solution dropwise, and stir and react at room temperature for about 1 - 2 h to obtain a crude solution of atosiban cis-dimer (α-Dimer). The HPLC purity of the crude product is 84.23%, the front impurity is 1.05%, and the back impurity is 0.98%.

[0121] The crude solution of atosiban cis-dimer (α-Dimer) is filtered through a 0.45 μm organic membrane, and purified and separated by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% acetic acid aqueous solution and acetonitrile for gradient elution, combine the fractions with a purity > 90% to obtain the dimer impurity combined sample. The combined sample is rotary evaporated and concentrated at 32 °C in a water bath to remove organic solvents, and then freeze-dried to obtain atosiban cis-dimer (α-Dimer):

[0122]

[0123] Weigh the weight of atosiban cis-dimer (α-Dimer) as 14.34 g, with a total yield of 72.13%, an HPLC purity of 97.96%, and the maximum single impurity of 0.47%.

[0124] Example 6. Synthesis of atosiban trans-dimer

[0125] Synthesis of atosiban linear peptide resin A

[0126] Weigh 10.5 g of Rink Amide AM resin (10 mmol, substitution value 0.95 mmol / g), add it to the polypeptide synthesis reaction column, add 100 ml of DMF to swell for 30 min, and drain. Wash twice with DMF and drain. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF. Weigh Fmoc-Gly-OH (5.95 g, 2.0 eq.), HOBt (2.97 g, 2.2 eq.) and dissolve them in 100 ml of DMF. Add DIC (4.7 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, add it to the synthesis reaction column, and react for 2 - 4 h under nitrogen protection at 25 ± 5 °C. Drain and wash 4 times with DMF.

[0127] Repeat the above steps to couple Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, and Mpa(Trt)-OH in sequence. Remove the Fmoc protecting group, then wash 4 times with DMF, 3 times with DCM, and 3 times with MeOH. After drying, 22.12 g of atosiban linear peptide resin A: Mpa(Trt)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Acm)-Pro-Orn(Boc)-Gly-Rink Amide AM resin is obtained.

[0128] Synthesis of atosiban linear peptide resin B

[0129] Weigh 10.5 g of Rink Amide AM resin (10 mmol, substitution value 0.95 mmol / g), add it to the polypeptide synthesis reaction column, add 100 ml of DMF to swell for 30 min, and drain. Wash twice with DMF and drain. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF. Weigh Fmoc-Gly-OH (5.95 g, 2.0 eq.), HOBt (2.97 g, 2.2 eq.) and dissolve them in 100 ml of DMF. Add DIC (4.7 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, add it to the synthesis reaction column, and react for 2 - 4 h under nitrogen protection at 25 ± 5 °C. Drain and wash 4 times with DMF.

[0130] Repeat the above steps to couple Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, and Mpa(Acm)-OH in sequence. Remove the Fmoc protecting group, and then wash with DMF 4 times, DCM 3 times, and MeOH 3 times. After drying, 22.86 g of atosiban linear peptide resin B: Mpa(Acm)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-Rink Amide AM resin is obtained.

[0131] Synthesis of atosiban linear peptide A2

[0132] Weigh 22.12 g of the above atosiban peptide resin A and add it to 22.12 ml of cleavage reagent 2 (TFA / Tis / H2O / m-cresol = 93.5 / 3.0 / 2.5 / 2.0). Carry out the cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 2500 ml of ice-cold isopropyl ether for precipitation. The solid is precipitated, centrifuged, and washed to obtain 11.52 g of atosiban linear peptide A2: Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys(Acm)-Pro-Orn-Gly-NH2.

[0133] Synthesis of atosiban linear peptide B1

[0134] Weigh 22.86 g of the above atosiban linear peptide resin B and add it to 206.9 ml of cleavage reagent 1 [(TFA / Tis / H2O / m-cresol = 93.5 / 3.0 / 2.5 / 2.0), 2,2-dithiopyridine (4.41 g, 2 eq.)]. Carry out the cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of ice-cold isopropyl ether for precipitation. The solid is precipitated, centrifuged, and washed to obtain 10.69 g of atosiban linear peptide B1: Mpa(Acm)-D-Tyr(Et)-Ile-Thr-Asn-Cys(S-Pyr)-Pro-Orn-Gly-NH2, that is

[0135]

[0136] First cyclization

[0137] Weigh 11.52 g of the above-mentioned atosiban linear peptide A2, and add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution;

[0138] Weigh 10.69 g of atosiban linear peptide B1, and add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution;

[0139] Add the above-mentioned acetic acid aqueous solution of linear peptide A2 to the acetic acid aqueous solution of linear peptide B1, and stir and react at room temperature for 1 - 2 h to form intermediate H. After the reaction is completed, the crude intermediate H solution is filtered through a 0.45 μm organic membrane and purified and separated by reverse-phase liquid chromatography. The packing material is reverse-phase C18, the particle size is 8 μm, and the pore size is Use a gradient elution with 0.1% TFA aqueous solution and acetonitrile, and combine the fractions with a purity > 80% to obtain the atosiban dimer impurity intermediate H.

[0140]

[0141] Second cyclization

[0142] Dropwise add 25% ammonia water to the purified intermediate H solution to adjust the pH = 8.0 ± 0.5;

[0143] Slowly dropwise add 1.2 ml of 30% hydrogen peroxide solution, and stir and react at room temperature for about 1 - 2 h to obtain a crude solution of atosiban trans-dimer (β-Dimer). The crude HPLC purity is 76.25%, the front impurity is 0.85%, and the back impurity is 1.44%.

[0144] The crude solution of atosiban trans-dimer (β-Dimer) is filtered through a 0.45 μm organic membrane and purified and separated by reverse-phase liquid chromatography. The packing material is reverse-phase C18, the particle size is 8 μm, and the pore size is Use a gradient elution with 0.1% acetic acid aqueous solution and acetonitrile, and combine the fractions with a purity > 90% to obtain the dimer impurity sample. The sample is rotary evaporated and concentrated in a 32°C water bath to remove the organic solvent, and then freeze-dried to obtain atosiban trans-dimer (β-Dimer):

[0145]

[0146] Weigh the weight of atosiban trans-dimer (β-Dimer) as 11.98 g, the total yield is 60.25%, the HPLC purity is 98.13%, and the maximum single impurity is 0.64%.

[0147] Example 7. Synthesis of terlipressin cis-dimer

[0148] Synthesis of terlipressin linear peptide resin A

[0149] Weigh 25.6 g of Rink Amide MBHA resin (20 mmol, substitution value 0.78 mmol / g), add it to a peptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and drain. Wash twice with DMF and drain. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF. Weigh Fmoc-Gly-OH (11.90 g, 2.0 eq.), HOBt (5.94 g, 2.2 eq.) and dissolve them in 100 ml of DMF. Add DIC (9.5 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, then add it to the synthesis reaction column and react for 2 - 4 h under nitrogen protection at 25 ± 5 °C. Drain and wash 4 times with DMF.

[0150] Repeat the above steps to couple Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH and Fmoc-Gly-OH in sequence. Remove the Fmoc protecting group, then wash 4 times with DMF, 3 times with DCM, 3 times with MeOH, and after drying, obtain 64.86 g of terlipressin linear peptide resin A: H-Gly-Gly-Gly-Cys(Trt)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-Lys(Boc)-Gly-Rink Amide MBHA resin.

[0151] Synthesis of terlipressin linear peptide A1

[0152] Weigh 32.43 g of the above terlipressin linear peptide resin A, add it to 312.6 ml of cleavage reagent 1 [(TFA / Tis / Phenol / H2O / m-cresol = 94 / 2 / 2 / 1 / 1), 4,4'-dithiodipyridine (4.41 g, 2 eq.)], and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of ice-cold isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, obtain 19.24 g of terlipressin linear peptide A1:

[0153]

[0154] Weigh 32.43 g of the above-mentioned terlipressin peptide resin A and add it to 312.6 ml of cleavage reagent 2 (TFA / Tis / Phenol / Mpr / m-cresol = 94 / 2 / 2 / 1 / 1). Carry out the cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of ice-cold isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, obtain 19.06 g of terlipressin linear peptide A2: H-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys(Acm)-Pro-Lys-Gly-NH2.

[0155] First cyclization

[0156] Weigh 19.24 g of the above-mentioned terlipressin linear peptide A1 and add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution.

[0157] Weigh 19.06 g of terlipressin linear peptide A2 and add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution.

[0158] Add the above-mentioned acetic acid aqueous solution of linear peptide A1 to the acetic acid aqueous solution of linear peptide A2, and stir at room temperature for 1 - 2 h to form intermediate I. After the reaction is completed, filter the crude intermediate I solution through a 0.45 μm organic membrane and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% TFA aqueous solution and acetonitrile for gradient elution, combine the fractions with a purity > 80% to obtain the terlipressin dimer impurity intermediate I.

[0159]

[0160] Second cyclization

[0161] Add 400 ml of acetic acid to the purified intermediate C solution and stir evenly.

[0162] Slowly add the iodine ethanol solution, keep the color of the solution light yellow and not fade, stir at room temperature for about 16 - 20 h, and after the reaction is completed, add ascorbic acid (Vc) to quench the reaction to obtain a crude solution of terlipressin cis-dimer (α-Dimer). The HPLC purity of the crude product is 86.92%, the front impurity is 2.56%, and the back impurity is 1.42%.

[0163] Filter the crude solution of terlipressin cis-dimer (α-Dimer) through a 0.45 μm organic membrane and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Gradient elution was carried out using 0.1% aqueous acetic acid solution and acetonitrile. The fractions with a purity > 90% were combined to obtain the dimer impurity sample. The sample was rotary evaporated and concentrated in a 32°C water bath to remove the organic solvent, and then lyophilized to obtain terlipressin cis-dimer (α-Dimer):

[0164]

[0165] The weight of terlipressin cis-dimer (α-Dimer) was weighed as 16.30 g, the total yield was 66.40%, the HPLC purity was 97.25%, and the maximum single impurity was 0.89%.

[0166] Example 8. Synthesis of terlipressin trans-dimer

[0167] Synthesis of terlipressin linear peptide resin A

[0168] Weigh 12.8 g of Rink Amide MBHA resin (10 mmol, substitution value 0.78 mmol / g), add it to the polypeptide synthesis reaction column, add 100 ml of DMF to swell for 30 min, and drain. Wash twice with DMF and drain. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF. Weigh Fmoc-Gly-OH (5.95 g, 2.0 eq.), HOBt (2.97 g, 2.2 eq.) and dissolve them in 100 ml of DMF. Add DIC (4.7 ml, 3.0 eq.) at 0 - 10°C to activate for 4 min, add it to the synthesis reaction column, and react for 2 - 4 h under nitrogen protection at 25 ± 5°C. Drain and wash 4 times with DMF.

[0169] Repeat the above steps to couple Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH and Fmoc-Gly-OH in sequence. Remove the Fmoc protecting group, and then wash 4 times with DMF, 3 times with DCM, and 3 times with MeOH. After drying, 32.66 g of terlipressin linear peptide resin A was obtained: H-Gly-Gly-Gly-Cys(Trt)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-Lys(Boc)-Gly-Rink Amide MBHA resin.

[0170] Synthesis of Terlipressin Linear Peptide Resin B

[0171] Weigh 12.8 g of Rink Amide MBHA resin (20 mmol, substitution value 0.78 mmol / g), add it to the polypeptide synthesis reaction column, add 100 ml of DMF to swell for 30 min, and drain. Wash twice with DMF and drain. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF. Weigh Fmoc-Gly-OH (5.95 g, 2.0 eq.), HOBt (2.97 g, 2.2 eq.) and dissolve them in 100 ml of DMF. Add DIC (4.7 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, then add it to the synthesis reaction column and react for 2 - 4 h under nitrogen protection at 25 ± 5 °C. Drain and wash 4 times with DMF.

[0172] Repeat the above steps to couple Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH and Fmoc-Gly-OH in sequence. Remove the Fmoc protecting group, then wash 4 times with DMF, 3 times with DCM, 3 times with MeOH, and after drying, obtain 33.08 g of Terlipressin Linear Peptide Resin B: H-Gly-Gly-Gly-Cys(Acm)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Lys(Boc)-Gly-Rink Amide MBHA resin.

[0173] Synthesis of Terlipressin Linear Peptide A1

[0174] Weigh 32.66 g of the above-mentioned Terlipressin Linear Peptide Resin A, add it to 312.6 ml of cleavage reagent 1 [(TFA / Tis / Phenol / H2O / m-cresol = 94 / 2 / 2 / 1 / 1), 4,4'-dithiodipyridine (4.41 g, 2 eq.)], and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, obtain 18.82 g of Terlipressin Linear Peptide A1:

[0175]

[0176] Synthesis of Terlipressin Linear Peptide B2

[0177] Weigh 33.08 g of the above-mentioned terlipressin peptide resin B containing aa1 (Acm) and aa2 (Trt), add it to 33.06 ml of cleavage reagent 2 (TFA / Tis / Phenol / Mpr / m-cresol = 94 / 2 / 2 / 1 / 1), and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and wash to obtain 18.66 g of terlipressin linear peptide B2: H-Gly-Gly-Gly-Cys(Acm)-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2.

[0178] First Cyclization

[0179] Weigh 18.82 g of the above-mentioned terlipressin linear peptide A1, add 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution;

[0180] Weigh 18.66 g of terlipressin linear peptide B2, add 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution;

[0181] Add the above-mentioned acetic acid aqueous solution of linear peptide A1 to the acetic acid aqueous solution of linear peptide B2, stir at room temperature for 1 - 2 h to form intermediate J. After the reaction is completed, filter the crude solution of intermediate J through a 0.45 μm organic membrane, and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% TFA aqueous solution and acetonitrile for gradient elution, combine the fractions with a purity > 80%, and obtain the terlipressin dimer impurity intermediate J.

[0182]

[0183] After the reaction is completed, purify the solution of intermediate J.

[0184] Second Cyclization

[0185] Add 400 ml of acetic acid to the purified solution of intermediate J and stir evenly;

[0186] Slowly add an iodine ethanol solution, keep the color of the solution light yellow and not fade, stir at room temperature for about 16 - 20 h, and add ascorbic acid (Vc) to quench the reaction after the reaction is completed to obtain a crude solution of terlipressin trans-dimer (β-Dimer). The HPLC purity of the crude product is 75.50%, the front impurity is 2.16%, and the back impurity is 0.96%.

[0187] The crude solution of terlipressin trans-dimer (β-Dimer) was filtered through a 0.45 μm organic membrane and purified and separated by reverse-phase liquid chromatography. The packing material was reverse-phase C18 with a particle size of 8 μm and a pore size of Gradient elution was carried out using 0.1% aqueous acetic acid solution and acetonitrile. The fractions with a purity > 90% were combined to obtain the dimer impurity sample. The sample was concentrated by rotary evaporation in a 32°C water bath to remove the organic solvent, and after freeze-drying, terlipressin trans-dimer (β-Dimer) was obtained:

[0188]

[0189] The weight of terlipressin trans-dimer (β-Dimer) was weighed as 13.47 g, the total yield was 54.87%, the HPLC purity was 96.89%, and the maximum single impurity was 1.00%.

[0190] Example 9: Synthesis of desmopressin cis-dimer

[0191] Synthesis of desmopressin linear peptide resin B

[0192] Weigh 25.6 g of Rink Amide MBHA resin (20 mmol, substitution value 0.78 mmol / g), add it to a polypeptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and drain. Wash twice with DMF and drain. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF. Weigh Fmoc-Gly-OH (11.90 g, 2.0 eq.), HOBt (5.94 g, 2.2 eq.) and dissolve them in 200 ml of DMF. Add DIC (9.5 ml, 3.0 eq.) at 0 - 10°C to activate for 4 min, add it to the synthesis reaction column, and react for 2 - 4 h under nitrogen protection at 25 ± 5°C. Drain and wash 4 times with DMF.

[0193] Repeat the above steps to couple Fmoc-D-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH and Mpr(Acm)-OH in sequence. Remove the Fmoc protecting group, then wash 4 times with DMF, 3 times with DCM, 3 times with MeOH, and after drying, 60.24 g of desmopressin linear peptide resin B: Mpr(Acm)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Rink Amide MBHA resin was obtained.

[0194] Synthesis of desmopressin linear peptide B1

[0195] Weigh 30.12 g of the above-mentioned desmopressin linear peptide resin B, add it to 301.2 ml of cleavage reagent 1 [(TFA / Tis / H2O / cresol = 93 / 3 / 3 / 1), pyridine disulfide (5.05 g, 2 eq.)], and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter, and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and wash to obtain 10.89 g of desmopressin linear peptide B1:

[0196]

[0197] Synthesis of desmopressin linear peptide B2

[0198] Weigh 30.12 g of the above-mentioned desmopressin linear peptide resin B, add it to 301.2 ml of cleavage reagent 2 (TFA / Tis / Mpr / cresol = 93 / 3 / 3 / 1), and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter, and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and wash to obtain 10.07 g of desmopressin linear peptide B2: Mpr(Acm)-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2.

[0199] First cyclization

[0200] Weigh 10.89 g of the above-mentioned desmopressin linear peptide B1, add it to 2000 ml of purified water and stir to dissolve, and add hydrochloric acid dropwise to adjust the pH = 4 - 6;

[0201] Weigh 10.07 g of the above-mentioned desmopressin linear peptide B2, add it to 2000 ml of purified water and stir to dissolve, and add hydrochloric acid dropwise to adjust the pH = 4 - 6;;

[0202] Add the above-mentioned aqueous hydrochloric acid solution of linear peptide B1 to the aqueous hydrochloric acid solution of linear peptide B2, and stir at room temperature for 1 - 2 h to form intermediate K. After the reaction is completed, filter the crude intermediate K solution through a 0.45 μm organic membrane, and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a gradient elution with 0.1% aqueous TFA solution and acetonitrile, combine the fractions with a purity > 80%, and obtain the desmopressin dimer impurity intermediate K.

[0203]

[0204] Second cyclization

[0205] Add 25% ammonia water dropwise to the purified intermediate K solution to adjust the pH to 8.0 ± 0.5;

[0206] Slowly add 1.2 ml of 30% hydrogen peroxide solution dropwise, stir and react at room temperature for about 1 - 2 h to obtain a crude solution of desmopressin cis-dimer (α-Dimer). The HPLC purity of the crude product is 87.21%, the front impurity is 1.06%, and the rear impurity is 2.16%.

[0207] Filter the crude solution of desmopressin cis-dimer (α-Dimer) through a 0.45 μm organic membrane, and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Perform gradient elution using 0.1% aqueous acetic acid solution and acetonitrile, combine the fractions with a purity > 90% to obtain a dimer impurity combined sample. Concentrate the combined sample by rotary evaporation in a 32°C water bath to remove the organic solvent, and obtain desmopressin cis-dimer (α-Dimer) after freeze-drying:

[0208]

[0209] Weigh the desmopressin cis-dimer (α-Dimer) to be 14.29 g, with a total yield of 66.82%, an HPLC purity of 98.17%, and the maximum single impurity of 0.73%.

[0210] Example 10, Synthesis of desmopressin trans-dimer

[0211] Synthesis of desmopressin linear peptide resin A

[0212] Weigh 12.8 g of Rink Amide MBHA resin (10 mmol, substitution value 0.78 mmol / g), add it to a polypeptide synthesis reaction column, add 100 ml of DMF to swell for 30 min, and drain. Wash with DMF twice and drain. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash with DMF 6 times. Weigh Fmoc-Gly-OH (5.95 g, 2.0 eq.), HOBt (2.97 g, 2.2 eq.) and dissolve them in 100 ml of DMF. Add DIC (4.7 ml, 3.0 eq.) at 0 - 10°C to activate for 4 min, add it to the synthesis reaction column, and react under nitrogen protection at 25 ± 5°C for 2 - 4 h. Drain and wash with DMF 4 times.

[0213] Repeat the above steps to couple Fmoc-D-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, and Mpr(Trt)-OH in sequence. Remove the Fmoc protecting group, and then wash with DMF 4 times, DCM 3 times, and MeOH 3 times. After drying, 28.85 g of desmopressin linear peptide resin A: Mpr(Trt)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-Arg(Pbf)-Gly-Rink Amide MBHA resin is obtained.

[0214] Synthesis of desmopressin linear peptide resin B

[0215] Weigh 12.8 g of Rink Amide MBHA resin (10 mmol, substitution value 0.78 mmol / g), add it to the polypeptide synthesis reaction column, add 100 ml of DMF to swell for 30 min, and drain. Wash with DMF twice and drain again. Add 20% PIPE / DMF to remove the Fmoc protecting group twice, with the deprotection times being 5 min + 10 min respectively, and wash with DMF 6 times. Weigh Fmoc-Gly-OH (5.95 g, 2.0 eq.), HOBt (2.97 g, 2.2 eq.) and dissolve them in 100 ml of DMF. Add DIC (4.7 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, and add it to the synthesis reaction column. React under nitrogen protection at 25 ± 5 °C for 2 - 4 h. Drain, and wash with DMF 4 times.

[0216] Repeat the above steps to couple Fmoc-D-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, and Mpr(Acm)-OH in sequence. Remove the Fmoc protecting group, and then wash with DMF 4 times, DCM 3 times, and MeOH 3 times. After drying, 29.82 g of desmopressin linear peptide resin B: Mpr(Acm)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Rink Amide MBHA resin is obtained.

[0217] Synthesis of desmopressin linear peptide A2

[0218] Weigh 28.85 g of the above-mentioned desmopressin linear peptide resin A, add it to 288.5 ml of cleavage reagent 2 (TFA / Mpr / H2O / m-cresol = 93 / 3 / 3 / 1), and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of ice-cold isopropyl ether for precipitation, precipitate the solid, centrifuge, and wash to obtain 12.25 g of desmopressin linear peptide A2: Mpr-Tyr-Phe-Gln-Asn-Cys(Acm)-Pro-Arg-Gly-NH2.

[0219] Synthesis of desmopressin linear peptide B1

[0220] Weigh 33.08 g of the above-mentioned desmopressin peptide resin B, add it to 33.06 ml of cleavage reagent 1 [(TFA / Tis / H2O / m-cresol = 93 / 3 / 3 / 1), pyridine disulfide (5.05 g, 2 eq.)], and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of ice-cold isopropyl ether for precipitation, precipitate the solid, centrifuge, and wash to obtain 11.83 g of desmopressin linear peptide B1:

[0221]

[0222] First cyclization

[0223] Weigh 12.25 g of the above-mentioned desmopressin linear peptide A2, add it to 2000 ml of purified water and stir to dissolve, then add hydrochloric acid dropwise to adjust the pH to 4 - 6;

[0224] Weigh 11.83 g of desmopressin linear peptide B1, add it to 2000 ml of purified water and stir to dissolve, then add hydrochloric acid dropwise to adjust the pH to 4 - 6;

[0225] Add the above-mentioned acetic acid aqueous solution of linear peptide A2 to the acetic acid aqueous solution of linear peptide B1, stir and react at room temperature for 1 - 2 h to form intermediate L. After the reaction is completed, filter the crude intermediate L solution through a 0.45 μm organic membrane, and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a gradient elution with 0.1% TFA aqueous solution and acetonitrile, combine the fractions with a purity > 80% to obtain the impurity intermediate L of terlipressin dimer.

[0226]

[0227] Second cyclization

[0228] Add 25% ammonia water dropwise to the purified intermediate L solution to adjust the pH to 8.0 ± 0.5;

[0229] Slowly add 1.2 ml of 30% hydrogen peroxide solution, stir and react at room temperature for about 1 - 2 h to obtain a crude solution of desmopressin trans-dimer (β-Dimer). The HPLC purity of the crude product is 72.98%, the front impurity is 1.48%, and the back impurity is 0.87%.

[0230] The crude solution of desmopressin trans-dimer (β-Dimer) is filtered through a 0.45 μm organic membrane and purified and separated by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Gradient elution is carried out using 0.1% acetic acid aqueous solution and acetonitrile. The fractions with a purity > 90% are combined to obtain a dimer impurity sample. The sample is rotary evaporated and concentrated in a 32°C water bath to remove the organic solvent, and then freeze-dried to obtain desmopressin trans-dimer (β-Dimer):

[0231]

[0232] Weigh the desmopressin trans-dimer (β-Dimer), and its weight is 12.46 g. The total yield is 58.27%, the HPLC purity is 98.56%, and the maximum single impurity is 0.55%.

[0233] Example 11, Synthesis of Ularitide Cis-Dimer

[0234] Synthesis of Ularitide Linear Peptide Resin A

[0235] Weigh 24.1 g of Wang resin (20 mmol, substitution value 0.83 mmol / g), add it to a polypeptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and then drain. Wash it twice with DMF and drain. Weigh Fmoc-Tyr(tBu)-OH (18.39 g, 2.0 eq.), HOBt (5.94 g, 2.2 eq.) and DMAP (0.49 g, 0.2 eq.), dissolve them in 200 ml of DMF, add DIC (9.5 ml, 3.0 eq.) at 0 - 10°C to activate for 4 min, add it to the synthesis reaction column, and react under nitrogen protection at 25 ± 5°C for 2 - 4 h. Drain, and wash 4 times with DMF. Add 20% PIPE / DMF to remove the Fmoc protection twice, and the deprotection times are 5 min + 10 min respectively. Wash 6 times with DMF.

[0236] Repeat the above steps to sequentially couple Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH and Fmoc-Thr(tBu)-OH. Remove the Fmoc protecting group, and then wash with DMF 4 times, DCM 3 times, and MeOH 3 times. After drying, 128.80 g of the linear peptide resin A of ulinastatin: H-Thr(tBu)-Ala-Pro-Arg(Pbf)-Ser(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gly-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Ac m)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-Wang resin is obtained.

[0237] Synthesis of the linear peptide A1 of ulinastatin

[0238] Weigh 64.40 g of the above-mentioned ulinastatin peptide resin A, add it to 644.0 ml of cleavage reagent 1 [(TFA / Tis / Phenol / m-cresol = 95 / 2 / 2 / 1), 2,2-dithiopyridine (4.41 g, 2 eq.)], and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter, and wash the resin with 100 ml of TFA. Add the filtrate to 6000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, obtain 38.72 g of ulinastatin linear peptide A1: H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys(S-Pyr)-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys(Acm)-Asn-Ser-Phe-Arg-Tyr-OH, that is

[0239]

[0240] H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys(Acm)-Asn-Ser-Phe-Arg-Tyr-OH

[0241] Synthesis of ulinastatin linear peptide A2

[0242] Weigh 64.40 g of the above-mentioned ulinastatin peptide resin A, add it to 312.6 ml of cleavage reagent 2 (TFA / Tis / Phenol / m-cresol = 95 / 2 / 2 / 1), and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter, and wash the resin with 100 ml of TFA. Add the filtrate to 6000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, obtain 37.98 g of ulinastatin linear peptide A2: H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys(Acm)-Asn-Ser-Phe-Arg-Tyr-OH.

[0243] First cyclization

[0244] Weigh 38.72 g of the above-mentioned ulinastatin linear peptide A1, add 2000 ml of purified water and stir to dissolve, and dropwise add phosphoric acid to adjust the pH = 4.0 - 6.0;

[0245] Weigh 37.98 g of the above-mentioned ulinastatin linear peptide A2, add 2000 ml of purified water and stir to dissolve, then add phosphoric acid dropwise to adjust the pH to 4.0 - 6.0;

[0246] Add the above-mentioned aqueous phosphoric acid solution of linear peptide A1 to the aqueous phosphoric acid solution of linear peptide A2, stir and react at room temperature for 1 - 2 h to form intermediate M. After the reaction is completed, the crude intermediate M solution is filtered through a 0.45 μm organic membrane and purified and separated by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a gradient elution with 0.1% aqueous TFA solution and acetonitrile, combine the fractions with a purity > 80% to obtain the ulinastatin dimer impurity intermediate M.

[0247]

[0248] Second cyclization

[0249] Add 400 ml of acetic acid to the purified intermediate M solution and stir evenly;

[0250] Slowly add the iodine ethanol solution, keep the color of the solution light yellow and not fade, stir and react at room temperature for about 16 - 20 h. After the reaction is completed, add ascorbic acid (Vc) to quench the reaction to obtain a crude solution of ulinastatin cis-dimer (α-Dimer). The crude HPLC purity is 79.26%, the front impurity is 0.96%, and the back impurity is 1.82%.

[0251] The crude solution of ulinastatin cis-dimer (α-Dimer) is filtered through a 0.45 μm organic membrane and purified and separated by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a gradient elution with 0.1% aqueous acetic acid solution and acetonitrile, combine the fractions with a purity > 90% to obtain the dimer impurity combined sample. The combined sample is rotary evaporated and concentrated in a 32°C water bath to remove the organic solvent, and then freeze-dried to obtain ulinastatin cis-dimer (α-Dimer):

[0252]

[0253] Weigh the weight of ulinastatin cis-dimer (α-Dimer) as 41.48 g, the total yield is 59.16%, the HPLC purity is 97.22%, and the maximum single impurity is 0.80%.

[0254] Example 12. Synthesis of ulinastatin trans-dimer

[0255] Synthesis of ulinastatin linear peptide resin A

[0256] Weigh 12.0 g of Wang resin (10 mmol, substitution value 0.83 mmol / g), add it to a peptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and drain. Wash twice with DMF and drain. Weigh Fmoc-Tyr(tBu)-OH (9.20 g, 2.0 eq.), HOBt (2.97 g, 2.2 eq.) and DMAP (0.25 g, 0.2 eq.) and dissolve them in 100 ml of DMF. Add DIC (4.7 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, then add it to the synthesis reaction column and react under nitrogen protection at 25 ± 5 °C for 2 - 4 h. Drain and wash 4 times with DMF. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF.

[0257] Repeat the above steps to couple Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH and Fmoc-Thr(tBu)-OH in sequence. Remove the Fmoc protecting group, then wash with DMF 4 times, DCM 3 times, and MeOH 3 times. After drying, 65.86 g of ulinastatin linear peptide resin A is obtained: H-Thr(tBu)-Ala-Pro-Arg(Pbf)-Ser(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gly-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Acm)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-Wang resin.

[0258] Synthesis of ulinastatin peptide resin B

[0259] Weigh 12.0 g of Wang resin (10 mmol, substitution value 0.83 mmol / g), add it to the peptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and then drain. Wash twice with DMF and drain. Weigh Fmoc-Tyr(tBu)-OH (9.20 g, 2.0 eq.), HOBt (2.97 g, 2.2 eq.) and DMAP (0.25 g, 0.2 eq.) and dissolve them in 100 ml of DMF. Add DIC (4.7 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, then add it to the synthesis reaction column and react for 2 - 4 h under nitrogen protection at 25 ± 5 °C. Drain and wash 4 times with DMF. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF.

[0260] Repeat the above steps to couple Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH and Fmoc-Thr(tBu)-OH in sequence. Remove the Fmoc protecting group, and then wash with DMF 4 times, DCM 3 times, and MeOH 3 times. After drying, 65.12 g of ulinastatin linear peptide resin B: H-Thr(tBu)-Ala-Pro-Arg(Pbf)-Ser(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Ser(tBu)-Ser(tBu)-Cys(Acm)-Phe-Gly-Gly-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-Wang resin is obtained.

[0261] Synthesis of Ulinastatin Linear Peptide A1

[0262] Weigh 65.86 g of the above-mentioned ulinastatin linear peptide resin A, add it to 651.2 ml of cleavage reagent 1 [(TFA / Tis / Phenol / m-cresol = 95 / 2 / 2 / 1), 2,2-dithiopyridine (4.41 g, 2 eq.)], and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 6000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, obtain 39.12 g of ulinastatin linear peptide A1: H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys(S-Pyr)-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys(Acm)-Asn-Ser-Phe-Arg-Tyr-OH, that is

[0263]

[0264] H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys(Acm)-Asn-Ser-Phe-Arg-Tyr-OH

[0265] Synthesis of ulinastatin linear peptide B2

[0266] Weigh 65.12 g of the above-mentioned ulinastatin peptide resin B, add it to 312.6 ml of cleavage reagent 2 (TFA / Tis / Phenol / m-cresol = 95 / 2 / 2 / 1), and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 6000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, obtain 37.55 g of ulinastatin linear peptide B2: H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys(Acm)-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH

[0267] First cyclization

[0268] Weigh 39.12 g of the above-mentioned ulinastatin linear peptide A1, add it to 2000 ml of purified water and stir to dissolve, and dropwise add phosphoric acid to adjust the pH = 4.0 - 6.0;

[0269] Weigh 37.55 g of the above-mentioned linear peptide B2 of ulinastatin, add 2000 ml of purified water and stir to dissolve it, and add phosphoric acid dropwise to adjust the pH to 4.0 - 6.0;

[0270] Add the above-mentioned aqueous phosphoric acid solution of linear peptide A1 to the aqueous phosphoric acid solution of linear peptide B2, and stir and react at room temperature for 1 - 2 h to form intermediate N. After the reaction is completed, the crude solution of intermediate N is filtered through a 0.45 μm organic membrane, and purified and separated by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a gradient elution with 0.1% aqueous TFA solution and acetonitrile, combine the fractions with a purity > 80%, and obtain the intermediate N of the dimer impurity of ulinastatin.

[0271]

[0272] Second cyclization

[0273] Add 400 ml of acetic acid to the purified intermediate N solution and stir evenly;

[0274] Slowly add an iodine ethanol solution, keep the color of the solution light yellow and not fade, stir and react at room temperature for about 16 - 20 h. After the reaction is completed, add ascorbic acid (Vc) to quench the reaction to obtain a crude solution of the trans-dimer (β-Dimer) of ulinastatin. The HPLC purity of the crude product is 69.04%, the front impurity is 1.28%, and the back impurity is 1.58%.

[0275] The crude solution of the trans-dimer (β-Dimer) of ulinastatin is filtered through a 0.45 μm organic membrane, and purified and separated by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a gradient elution with 0.1% aqueous acetic acid solution and acetonitrile, combine the fractions with a purity > 90%, and obtain the dimer impurity sample. The sample is concentrated by rotary evaporation in a 32°C water bath to remove the organic solvent, and freeze-dried to obtain the trans-dimer (β-Dimer) of ulinastatin:

[0276]

[0277] Weigh the weight of the trans-dimer (β-Dimer) of ulinastatin as 32.90 g, the total yield is 46.92%, the HPLC purity is 98.24%, and the maximum single impurity is 0.46%.

[0278] Example 13, Synthesis of the cis-dimer of somatostatin

[0279] Synthesis of the linear peptide resin A of somatostatin

[0280] Weigh 22.22 g of 2CTC resin (20 mmol, substitution value 0.90 mmol / g), add it to the polypeptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and then drain. Wash it twice with DMF and drain. Weigh Fmoc-Cys(Acm)-OH (33.18 g, 4.0 eq.), DIEA (31.02 g, 12.0 eq.) and DMAP (0.98 g, 0.4 eq.), add them to the synthesis reaction column, and react for 5 h under nitrogen protection at 25 ± 5 °C. Drain and wash 4 times with DMF.

[0281] Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF. Weigh Fmoc-Ser(tBu)-OH (15.35 g, 2.0 eq.) and HOBt (5.94 g, 2.2 eq.), dissolve them in 100 ml of DMF, add DIC (9.5 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, add it to the synthesis reaction column, and react for 2 - 4 h under nitrogen protection at 25 ± 5 °C. Drain and wash 4 times with DMF.

[0282] Repeat the above steps to couple Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH in sequence. Remove the Fmoc protecting group, and then wash 4 times with DMF, 3 times with DCM, and 3 times with MeOH. After drying, 76.84 g of somatostatin linear peptide resin A: H-Ala-Gly-Cys(Trt)-Lys(Boc)-Asn(Trt)-Phe-Phe-Trp(Boc)-Lys(Boc)-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Cys(Acm)-2CTC resin is obtained.

[0283] Synthesis of somatostatin linear peptide A1

[0284] Weigh 38.42 g of the above somatostatin linear peptide resin A, add it to 384.2 ml of cleavage reagent 1 [(TFA / Tis / H2O = 95.0 / 2.5 / 2.5), 2,2-dithiodipyridine (4.41 g, 2 eq.)], and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, obtain 28.86 g of somatostatin linear peptide A1: H-Ala-Gly-Cys(S-Pyr)-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys(Acm)-OH, that is

[0285] Synthesis of somatostatin linear peptide A2

[0286] Weigh 38.42 g of the above somatostatin linear peptide resin A, add it to 384.2 ml of cleavage reagent 2 (TFA / Tis / H2O = 95.0 / 2.5 / 2.5), and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, obtain 27.23 g of somatostatin linear peptide A2: H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys(Acm)-OH.

[0287] First cyclization

[0288] Weigh 28.86 g of the above somatostatin linear peptide A1, add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution;

[0289] Weigh 27.23 g of somatostatin linear peptide A2, add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution;

[0290] Add the above acetic acid aqueous solution of linear peptide A1 to the acetic acid aqueous solution of linear peptide A2, stir and react at room temperature for 1 - 2 h to form intermediate O. After the reaction is completed, filter the crude intermediate O solution through a 0.45 μm organic membrane, and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% TFA aqueous solution and acetonitrile for gradient elution, combine the fractions with a purity > 80%, and obtain the somatostatin dimer impurity intermediate O.

[0291]

[0292] Second cyclization

[0293] Add 400 ml of acetic acid to the purified intermediate O solution and stir evenly.

[0294] Slowly add the iodoethanol solution, keep the color of the solution light yellow and not fade, stir and react at room temperature for about 16 - 20 h. After the reaction is completed, add ascorbic acid (Vc) to quench the reaction to obtain a crude solution of somatostatin cis - dimer (α - Dimer). The crude HPLC purity is 58.23%, the front impurity is 3.42%, and the back impurity is 4.52%.

[0295] The crude solution of somatostatin cis - dimer (α - Dimer) is filtered through a 0.45 μm organic membrane and purified and separated by reverse - phase liquid chromatography. The packing material is reverse - phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% aqueous acetic acid solution and acetonitrile for gradient elution, combine the fractions with a purity > 90% to obtain a dimer impurity combined sample. The combined sample is rotary - evaporated and concentrated in a 32°C water bath to remove the organic solvent, and then freeze - dried to obtain somatostatin cis - dimer (α - Dimer):

[0296]

[0297] Weigh the weight of somatostatin cis - dimer (α - Dimer) as 19.40 g, the total yield is 59.23%, the HPLC purity is 98.74%, and the maximum single impurity is 0.39%.

[0298] Example 14. Synthesis of somatostatin trans - dimer

[0299] Synthesis of somatostatin linear peptide resin A

[0300] Weigh 22.22 g of 2CTC resin (20 mmol, substitution value 0.90 mmol / g), add it to the polypeptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and drain. Then wash twice with DMF and drain. Weigh Fmoc - Cys(Acm)-OH (33.18 g, 4.0 eq.), DIEA (31.02 g, 12.0 eq.) and DMAP (0.98 g, 0.4 eq.), add them to the synthesis reaction column, and react under nitrogen protection at 25 ± 5°C for 5 h. Drain and wash 4 times with DMF.

[0301] Deprotect the Fmoc group twice by adding 20% PIPE / DMF, with the deprotection times being 5 min + 10 min respectively, and wash with DMF 6 times. Weigh Fmoc-Ser(tBu)-OH (15.35 g, 2.0 eq.), HOBt (5.94 g, 2.2 eq.), dissolve them in 100 ml of DMF, add DIC (9.5 ml, 3.0 eq.) at 0 - 10 °C for activation for 4 min, add it to the synthesis reaction column, and react for 2 - 4 h under nitrogen protection at 25 ± 5 °C. Drain, and wash with DMF 4 times.

[0302] Repeat the above steps to couple Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH in sequence. Deprotect the Fmoc protecting group, then wash with DMF 4 times, wash with DCM 3 times, wash with MeOH 3 times, and after drying, obtain 76.84 g of somatostatin linear peptide resin A: H-Ala-Gly-Cys(Trt)-Lys(Boc)-Asn(Trt)-Phe-Phe-Trp(Boc)-Lys(Boc)-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Cys(Acm)-2CTC resin.

[0303] Synthesis of somatostatin linear peptide resin B

[0304] Weigh 22.22 g of 2CTC resin (20 mmol, substitution value 0.90 mmol / g), add it to the polypeptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and drain. Then wash with DMF twice and drain. Weigh Fmoc-Cys(Trt)-OH (46.89 g, 4.0 eq.), DIEA (31.02 g, 12.0 eq.), and DMAP (0.98 g, 0.4 eq.), add them to the synthesis reaction column, and react for 5 h under nitrogen protection at 25 ± 5 °C. Drain, and wash with DMF 4 times.

[0305] Fmoc protection was removed twice by adding 20% PIPE / DMF, with the deprotection times being 5 min + 10 min respectively, and washed 6 times with DMF. Weigh Fmoc-Ser(tBu)-OH (15.35 g, 2.0 eq.), HOBt (5.94 g, 2.2 eq.) and dissolve them in 100 ml of DMF. Add DIC (9.5 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, then add it to the synthesis reaction column and react for 2 - 4 h under nitrogen protection at 25 ± 5 °C. Drain, and wash 4 times with DMF.

[0306] Repeat the above steps, and couple Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH in turn. Remove the Fmoc protecting group, then wash 4 times with DMF, 3 times with DCM, and 3 times with MeOH. After drying, 76.84 g of somatostatin linear peptide resin B: H-Ala-Gly-Cys(Acm)-Lys(Boc)-Asn(Trt)-Phe-Phe-Trp(Boc)-Lys(Boc)-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Cys(Trt)-2CTC resin was obtained.

[0307] Synthesis of somatostatin linear peptide A1

[0308] Weigh 38.42 g of the above somatostatin linear peptide resin A and add it to 384.2 ml of cleavage reagent 1 [(TFA / Tis / H2O = 95.0 / 2.5 / 2.5), 2,2-dithiopyridine (4.41 g, 2 eq.)], and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, 28.86 g of somatostatin linear peptide A1: H-Ala-Gly-Cys(S-Pyr)-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys(Acm)-OH was obtained, that is

[0309] Synthesis of somatostatin linear peptide B2

[0310] Weigh 37.52 g of the above-mentioned somatostatin linear peptide resin B and add it to 375.2 ml of cleavage reagent 2 (TFA / Tis / H2O = 95.0 / 2.5 / 2.5). Carry out the cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of frozen isopropyl ether for precipitation. The solid is precipitated, centrifuged, and washed to obtain 26.76 g of somatostatin linear peptide B2: H-Ala-Gly-Cys(Acm)-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH.

[0311] First cyclization

[0312] Weigh 28.86 g of the above-mentioned somatostatin linear peptide A1 and add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution.

[0313] Weigh 26.76 g of somatostatin linear peptide B2 and add it to 200 ml of acetic acid and 1800 ml of purified water to form a 10% acetic acid aqueous solution.

[0314] Add the above-mentioned acetic acid aqueous solution of linear peptide A1 to the acetic acid aqueous solution of linear peptide B2, stir and react at room temperature for 1 - 2 h to form intermediate P. After the reaction is completed, filter the crude intermediate P solution through a 0.45 μm organic membrane and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% TFA aqueous solution and acetonitrile for gradient elution, combine the fractions with a purity > 80% to obtain the somatostatin dimer impurity intermediate P.

[0315]

[0316] Second cyclization

[0317] Add 400 ml of acetic acid to the purified intermediate P solution and stir evenly.

[0318] Slowly add the iodine ethanol solution, keep the color of the solution light yellow and not fade, stir and react at room temperature for about 16 - 20 h. After the reaction is completed, add ascorbic acid (Vc) to quench the reaction to obtain a crude solution of somatostatin trans-dimer (β-Dimer). The HPLC purity of the crude product is 56.79%, the front impurity is 3.52%, and the back impurity is 2.12%.

[0319] Filter the crude solution of somatostatin trans-dimer (β-Dimer) through a 0.45 μm organic membrane and purify and separate it by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Gradient elution was performed using a 0.1% aqueous acetic acid solution and acetonitrile. Fractions with a purity > 90% were combined to obtain a dimer impurity sample. The sample was rotary evaporated and concentrated in a 32°C water bath to remove the organic solvent, and then freeze-dried to obtain somatostatin trans-dimer (β-Dimer):

[0320]

[0321] The weight of the somatostatin trans-dimer (β-Dimer) was weighed as 16.23 g, the total yield was 49.54%, the HPLC purity was 98.68%, and the maximum single impurity was 0.43%.

[0322] Comparative Example 1: Synthesis of Oxytocin Cis-Dimer

[0323] Synthesis of Oxytocin Linear Peptide Resin A

[0324] Weigh 25.6 g of Rink Amide MBHA resin (20 mmol, substitution value 0.78 mmol / g), add it to a polypeptide synthesis reaction column, add 200 ml of DMF to swell for 30 min, and drain. Wash twice with DMF and drain. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF. Weigh Fmoc-Gly-OH (11.90 g, 2.0 eq.), HOBt (5.94 g, 2.2 eq.) and dissolve them in 100 ml of DMF. Add DIC (9.5 ml, 3.0 eq.) at 0 - 10°C to activate for 4 min, add it to the synthesis reaction column, and react for 2 - 4 h under nitrogen protection at 25 ± 5°C. Drain and wash 4 times with DMF.

[0325] Repeat the above steps to sequentially couple Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH and Fmoc-Cys(Trt)-OH. Remove the Fmoc protecting group, and then wash 4 times with DMF, 3 times with DCM, and 3 times with MeOH. After drying, 63.86 g of oxytocin linear peptide resin A containing aa1(Trt) and aa2(Acm) was obtained: H-Cys(Trt)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-Leu-Gly-Rink Amide MBHA resin.

[0326] Synthesis of Oxytocin Linear Peptide A2

[0327] Weigh 63.86 g of the above-mentioned oxytocin linear peptide resin A, add it to 638.6 ml of a cleavage reagent (TFA / Tis / H2O = 95.0 / 2.5 / 2.5), and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 6500 ml of ice-cold isopropyl ether for precipitation, precipitate the solid, centrifuge, and wash to obtain 36.53 g of oxytocin linear peptide A2: H-Cys-Tyr-Ile-Gln-Asn-Cys(Acm)-Pro-Leu-Gly-NH2.

[0328] First cyclization

[0329] Weigh 36.53 g of the above-mentioned oxytocin linear peptide A2, add it to 2000 ml of purified water and stir to dissolve, then add 25% ammonia water dropwise to adjust the pH to 8.0 ± 0.5;

[0330] Slowly add 1.2 ml of 30% hydrogen peroxide solution, and stir and react at room temperature for about 1 - 2 h to form an oxytocin dimer impurity intermediate E.

[0331]

[0332] Second cyclization

[0333] Add 400 ml of acetic acid to the intermediate E solution and stir evenly;

[0334] Slowly add an iodine ethanol solution, keep the color of the solution light yellow and not fade, stir and react at room temperature for about 16 - 20 h, and after the reaction is completed, add ascorbic acid (Vc) to quench the reaction to obtain a crude solution of oxytocin cis-dimer (α-Dimer). The HPLC purity of the crude product is 63.11%, the front impurity is 4.86%, and the back impurity is 2.18%.

[0335] The crude solution of oxytocin cis-dimer (α-Dimer) is filtered through a 0.45 μm organic membrane and purified and separated by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% aqueous acetic acid solution and acetonitrile for gradient elution, combine the fractions with a purity > 90% to obtain a dimer impurity combined sample. The combined sample is rotary evaporated and concentrated at 32 °C in a water bath to remove the organic solvent, and after freeze-drying, oxytocin cis-dimer (α-Dimer) is obtained:

[0336]

[0337] Weigh the weight of oxytocin cis-dimer (α-Dimer) as 2.64 g, the total yield is 13.11%, the HPLC purity is 95.86%, and the maximum single impurity is 1.67%.

[0338] Synthesis of Oxytocin Trans-dimer, Comparative Example 2

[0339] Synthesis of Oxytocin Linear Peptide Resin A

[0340] Weigh 12.8 g of Rink Amide MBHA resin (10 mmol, substitution value 0.78 mmol / g), add it to the polypeptide synthesis reaction column, add 100 ml of DMF to swell for 30 min, and drain. Wash twice with DMF and drain. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF. Weigh Fmoc-Gly-OH (5.95 g, 2.0 eq.), HOBt (2.97 g, 2.2 eq.) and dissolve them in 50 ml of DMF. Add DIC (4.7 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, add it to the synthesis reaction column, and react for 2 - 4 h under nitrogen protection at 25 ± 5 °C. Drain and wash 4 times with DMF.

[0341] Repeat the above steps to couple Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH and Fmoc-Cys(Trt)-OH in sequence. Remove the Fmoc protecting group, then wash 4 times with DMF, 3 times with DCM, 3 times with MeOH, and after drying, obtain 33.28 g of oxytocin linear peptide resin A containing aa1(Trt) and aa2(Acm): H-Cys(Trt)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-Leu-Gly-Rink Amide MBHA resin.

[0342] Synthesis of Oxytocin Linear Peptide Resin B

[0343] Weigh 12.8 g of Rink Amide MBHA resin (10 mmol, substitution value 0.78 mmol / g), add it to a peptide synthesis reaction column, add 100 ml of DMF to swell for 30 min, and drain. Wash twice with DMF and drain. Weigh Fmoc-Gly-OH (5.95 g, 2.0 eq.), HOBt (2.97 g, 2.2 eq.) and dissolve them in 50 ml of DMF. Add DIC (4.7 ml, 3.0 eq.) at 0 - 10 °C to activate for 4 min, then add it to the synthesis reaction column and react for 2 - 4 h under nitrogen protection at 25 ± 5 °C. Drain and wash 4 times with DMF. Add 20% PIPE / DMF to remove the Fmoc protection twice, with the deprotection times being 5 min + 10 min respectively, and wash 6 times with DMF.

[0344] Repeat the above steps to couple Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH and Fmoc-Cys(Acm)-OH in sequence. Remove the Fmoc protecting group, then wash 4 times with DMF, 3 times with DCM, 3 times with MeOH, and after drying, obtain 31.56 g of oxytocin linear peptide resin B: H-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Rink Amide MBHA resin.

[0345] Synthesis of oxytocin linear peptide A2

[0346] Weigh 33.28 g of the above oxytocin peptide resin A, add it to 320.4 ml of cleavage reagent (TFA / Tis / H2O = 95.0 / 2.5 / 2.5), and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of ice-cold isopropyl ether for precipitation, precipitate the solid, centrifuge, and after washing, obtain 18.87 g of oxytocin linear peptide A2: H-Cys-Tyr-Ile-Gln-Asn-Cys(Acm)-Pro-Leu-Gly-NH2.

[0347] Synthesis of oxytocin linear peptide B2

[0348] Weigh 32.56 g of the above-mentioned oxytocin peptide resin B, add it to 327.8 ml of cleavage reagent (TFA / Tis / H2O = 95.0 / 2.5 / 2.5), and carry out a cleavage reaction at 25 ± 5 °C for 1 - 4 h. After the reaction is completed, filter and wash the resin with 100 ml of TFA. Add the filtrate to 3000 ml of frozen isopropyl ether for precipitation, precipitate the solid, centrifuge, and wash to obtain 17.89 g of oxytocin linear peptide B2: H-Cys(Acm)-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2.

[0349] First cyclization

[0350] Weigh 18.87 g of the above-mentioned oxytocin linear peptide A2 and 17.89 g of oxytocin linear peptide B2, add them to 2000 ml of purified water, stir to dissolve, and add 25% ammonia water dropwise to adjust the pH = 8.0 ± 0.5;

[0351] Slowly add 1.2 ml of 30% hydrogen peroxide solution, and stir and react at room temperature for about 1 - 2 h to form an oxytocin dimer impurity intermediate F.

[0352]

[0353] Second cyclization

[0354] Add 400 ml of acetic acid to the intermediate F solution and stir evenly;

[0355] Slowly add an iodine ethanol solution, keep the color of the solution light yellow and not fade, stir and react at room temperature for about 16 - 20 h. After the reaction is completed, add ascorbic acid (Vc) to quench the reaction to obtain a crude solution of oxytocin trans-dimer (β-Dimer). The HPLC purity of the crude product is 13.84%, the front impurity is 44.29%, and the back impurity is 0.86%. The retention time of the front impurity is the same as that of oxytocin cis-dimer (α-Dimer), indicating that during the first cyclization, a reaction occurs between linear peptide A2 and linear peptide A2 or between linear peptide B2 and linear peptide B2 to form oxytocin cis-dimer. Therefore, directional synthesis cannot be achieved.

[0356] The crude solution of oxytocin trans-dimer (β-Dimer) is filtered through a 0.45 μm organic membrane and purified and separated by reverse-phase liquid chromatography. The packing material is reverse-phase C18, with a particle size of 8 μm and a pore size of Use a 0.1% acetic acid aqueous solution and acetonitrile for gradient elution, combine the fractions with a purity > 90% to obtain a dimer impurity combined sample. The combined sample is rotary evaporated and concentrated in a 32 °C water bath to remove the organic solvent, and then freeze-dried to obtain oxytocin trans-dimer (β-Dimer):

[0357]

[0358] The weight of oxytocin trans-dimer (β-Dimer) was weighed as 1.80 g, the total yield was 8.92%, the HPLC purity was 97.15%, and the maximum single impurity was 1.13%.

[0359] Comparative Example 3, Synthesis of atosiban cis-dimer

[0360] Synthesis of atosiban linear peptide resin B

[0361] Weigh 10.6 g of Rink Amide AM resin (10 mmol, substitution value 0.95 mmol / g), add it to the polypeptide synthesis reaction column, swell it with 70 ml of DCM for 30 min, and drain. Wash it twice with DMF and drain. Add 20% PIP / DMF to deprotect Fmoc twice (the times are 5 min + 10 min respectively), and wash it 6 times with DMF.

[0362] Weigh 8.92 g of Fmoc-Gly-OH (3.0 eq) and 5.40 g of HOBt (4.0 eq), dissolve them in 40 ml of DMF, add 6.2 ml of DIC (4.0 eq) at 0 °C to 10 °C, activate for 5 min, add it to the synthesis reaction column, stir and react at 30 °C ± 3 °C for 2 - 3 h, and the ninhydrin test shows negative. Drain, and wash it 3 times with DMF. Add 20% PIP / DMF to deprotect Fmoc twice (the times are 5 min + 10 min respectively), and wash it 6 times with DMF.

[0363] Repeat the above steps, and couple Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, and Mpa(Acm)-OH in sequence. After washing and drying, 19.8 g of atosiban linear peptide resin B: Mpa(Acm)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-Rink Amide AM resin is obtained.

[0364] Synthesis of atosiban linear peptide B2

[0365] Add the above-mentioned peptide resin B to 200 ml of cleavage reagent (TFA / Tis / Phenol / Mpr = 92.5 / 2.5 / 2.5 / 2.5)), and carry out the cleavage reaction at 25°C ± 5°C for 2 h. After the reaction is completed, filter, add the filtrate to 1.6 L of methyl tert-butyl ether at 0°C ± 5°C for precipitation, centrifuge, and wash to obtain atosiban linear peptide B2: Mpa(Acm)-D-Tyr(Et)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH2.

[0366] First cyclization

[0367] Add 5 L of purified water, stir to dissolve, adjust the pH value of the solution to 7.0 - 7.5 with dilute ammonia water, add 10 ml of hydrogen peroxide solution, and stir and react for 1 - 2 h to obtain atosiban dimer impurity intermediate G:

[0368]

[0369] Second cyclization

[0370] Slowly add 6 mol / L iodine / methanol solution to the crude solution of atosiban intermediate G until the reaction solution turns reddish-brown and does not fade after stirring for 3 min. Continue to stir and react for 4 - 6 h. If the color of the solution fades during the reaction, add iodine / methanol solution to ensure that the reaction solution remains reddish-brown. After the reaction is completed, add vitamin C / methanol solution to quench the reaction until the reaction solution becomes colorless. The crude solution of atosiban cis-dimer (α-Dimer) can be obtained, with a crude HPLC purity of 42.53%, a front impurity of 8.26%, and a back impurity of 2.68%.

[0371] Filter with a 0.45 μm filter membrane and purify and separate by semi-preparative reversed-phase high-performance liquid chromatography. The filler used is C18, with a particle size of 10 μm and a pore size of The chromatographic column specifications are 50 mm * 250 mm. Use 0.3% acetic acid / purified water as mobile phase A and acetonitrile (chromatographically pure) as mobile phase B. The detection wavelength is 230 nm. Equilibrate with 5% mobile phase B for 10 min, increase to 34% within 1 min, and increase from 34% to 50% within 60 min until the elution is completed. Collect the components from 37 min to 41 min. Concentrate the collected qualified sample solution at 30°C ± 5°C to remove acetonitrile, and freeze-dry to obtain 1.03 g of atosiban cis-dimer (α-Dimer):

[0372]

[0373] Weigh the atosiban cis-dimer (α-Dimer) with a weight of 1.03 g, the yield is 5.18%, the HPLC purity is 98.39%, and the maximum single impurity is 0.96%.

[0374] Comparative Example 4, Synthesis of Atosiban Trans-Dimer

[0375] Synthesis of Atosiban Linear Peptide Resin A

[0376] Weigh 12.5 g of Rink Amide AM resin (10 mmol, substitution value 0.80 mmol / g), add it to a peptide synthesis reaction column, swell it with 70 ml of DCM for 30 min, and drain. Wash it twice with DMF and drain. Add 20% PIP / DMF to deprotect Fmoc twice (times are 5 min + 10 min respectively), and wash it 6 times with DMF.

[0377] Weigh 8.92 g of Fmoc-Gly-OH (3.0 eq) and 5.40 g of HOBt (4.0 eq), dissolve them in 40 ml of DMF, add 6.2 ml of DIC (4.0 eq) at 0 °C - 10 °C, activate for 5 min, add it to the synthesis reaction column, stir and react at 30 °C ± 3 °C for 2 - 3 h, and the ninhydrin test shows negative. Drain, and wash it 3 times with DMF. Add 20% PIP / DMF to deprotect Fmoc twice (times are 5 min + 10 min respectively), and wash it 6 times with DMF.

[0378] Repeat the above steps to couple Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, and Mpa(Trt)-OH in sequence. After washing and drying, 20.5 g of Atosiban linear peptide resin A: Mpa(Trt)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Acm)-Pro-Orn(Boc)-Gly-Rink Amide AM resin is obtained.

[0379] Synthesis of Atosiban Peptide Resin B

[0380] Weigh 10.6 g of Rink Amide AM resin (10 mmol, substitution value 0.95 mmol / g), add it to a peptide synthesis reaction column, swell it with 70 ml of DCM for 30 min, and drain. Wash it twice with DMF and drain. Add 20% PIP / DMF to deprotect Fmoc twice (times are 5 min + 10 min respectively), and wash it 6 times with DMF.

[0381] Weigh 8.92 g of Fmoc-Gly-OH (3.0 eq) and 5.40 g of HOBt (4.0 eq), dissolve them in 40 ml of DMF, add 6.2 ml of DIC (4.0 eq) at 0 °C - 10 °C, activate for 5 min, add it to the synthesis reaction column, stir and react at 30 °C ± 3 °C for 2 - 3 h, and the ninhydrin test shows negative. Drain, wash 3 times with DMF. Add 20% PIP / DMF to deprotect Fmoc twice (the times are 5 min + 10 min respectively), and wash 6 times with DMF.

[0382] Repeat the above steps, and couple Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, Mpa(Acm)-OH in sequence. After washing and drying, 19.8 g of atosiban linear peptide resin B: Mpa(Acm)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-Rink Amide AM resin is obtained.

[0383] Synthesis of atosiban linear peptide A2

[0384] Add the above linear peptide resin A to 200 ml of cleavage reagent (TFA / Tis / H2O / cresol = 93.5 / 3.0 / 2.5 / 2.0), and carry out a cleavage reaction at 25 °C ± 5 °C for 2 h. After the reaction is completed, filter, add the filtrate to 1.6 L of methyl tert-butyl ether at 0 °C ± 5 °C for sedimentation, centrifuge, and wash to obtain atosiban linear peptide A2: and Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys(Acm)-Pro-Orn-Gly-NH2.

[0385] Synthesis of atosiban linear peptide B2

[0386] Add the above linear peptide resin B to 200 ml of cleavage reagent (TFA / Tis / H2O / cresol = 93.5 / 3.0 / 2.5 / 2.0), and carry out a cleavage reaction at 25 °C ± 5 °C for 2 h. After the reaction is completed, filter, add the filtrate to 1.6 L of methyl tert-butyl ether at 0 °C ± 5 °C for sedimentation, centrifuge, and wash to obtain atosiban linear peptide B2: Mpa(Acm)-D-Tyr(Et)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH2.

[0387] First cyclization:

[0388] Add the above linear peptide A2 and linear peptide B2 to 4 L of purified water, stir to dissolve, adjust the pH value of the solution to 7.0 - 7.5 with dilute ammonia water, add 10 ml of hydrogen peroxide solution, and stir and react for 1 - 2 h. Obtain the atosiban dimer impurity intermediate H:

[0389]

[0390] Second cyclization:

[0391] Slowly add 6 mol / L iodine / methanol solution to the crude solution of atosiban intermediate H until the reaction solution turns reddish-brown and does not fade after stirring for 3 min. Continue to stir and react for 4 - 6 h. If the color of the solution fades during the reaction, add iodine / methanol solution to ensure that the reaction solution remains reddish-brown. After the reaction is completed, add vitamin C / methanol solution to quench the reaction until the reaction solution becomes colorless. The crude solution of atosiban impurity trans-dimer (β-Dimer) can be obtained. The crude HPLC purity is 13.84%, the front impurity is 2.63%, the back impurity is 44.29%, and the retention time is the same as that of atosiban cis-dimer (α-Dimer), indicating that during the first cyclization, a reaction occurs between linear peptide A2 and linear peptide A2 or between linear peptide B2 and linear peptide B2 to form atosiban cis-dimer. Therefore, directional synthesis cannot be achieved.

[0392] Filter with a 0.45 μm filter membrane and purify and separate by semi-preparative reverse-phase high-performance liquid chromatography. The filler used is C18, with a particle size of 10 μm, and the pore size chromatographic column specification is 50 mm * 250 mm. Use 0.3% acetic acid / purified water as mobile phase A and acetonitrile (chromatographically pure) as mobile phase B. The detection wavelength is 230 nm. Equilibrate with 5% mobile phase B for 10 min, increase to 34% within 1 min, and increase from 34% to 50% within 60 min until the elution is completed. Collect the components at 37 min - 41 min. Concentrate the collected qualified sample solution at 30°C ± 5°C to remove acetonitrile, and obtain 0.62 g of atosiban trans-dimer (β-Dimer) after freeze-drying:

[0393]

[0394] Weigh the atosiban trans-dimer (β-Dimer), with a weight of 0.62 g, a yield of 3.12%, an HPLC purity of 98.05%, and the maximum single impurity of 1.04%.

Claims

1. A method for the directed synthesis of a polypeptide dimer, characterized in that, It includes the following steps: (1) Prepare linear peptide resin A containing aa1(Trt) and aa2(Acm), and linear peptide resin B containing aa1(Acm) and aa2(Trt); (2) Use cleavage reagent 1 to cleave resins A and B to obtain linear peptide A1 containing aa1(activating group) and aa2(Acm), and linear peptide B1 containing aa1(Acm) and aa2(activating group); use cleavage reagent 2 to cleave resins A and B to obtain linear peptide A2 containing aa1 and aa2(Acm), and linear peptide B2 containing aa1(Acm) and aa2; (3) React the linear peptide containing the activating group and the linear peptide without the activating group in an acidic solution for the first cyclization to form a crude solution of polypeptide dimer impurity intermediate. After filtering the crude solution, purify and separate it by reverse-phase liquid chromatography, perform gradient elution using 0.1% aqueous TFA solution and acetonitrile, and combine the fractions with a purity > 80% to obtain the polypeptide dimer intermediate; (4) Remove Acm and perform the second cyclization to obtain a crude solution of cis- or trans-polypeptide dimer. After filtration, purify and separate it by reverse-phase liquid chromatography, perform gradient elution using 0.1% aqueous acetic acid solution and acetonitrile, combine the fractions with a purity > 90%, concentrate by rotary evaporation in a water bath and freeze-dry to obtain cis- or trans-polypeptide dimer; Among them, the chromatographic packing materials in steps (3) and (4) are reverse-phase C18 with a particle size of 8 μm and a pore size of Wherein, the aa1 and aa2 are selected from Cys and Mpa; Wherein, the polypeptide is selected from oxytocin, atosiban, octreotide, terlipressin, desmopressin, ulinastatin, and somatostatin; Wherein, when synthesizing the cis-polypeptide dimer, the linear peptide containing the activating group and the linear peptide without the activating group in step (3) are linear peptides A1 and A2, or linear peptides B1 and B2; when synthesizing the trans-polypeptide dimer, the linear peptide containing the activating group and the linear peptide without the activating group in step (3) are linear peptides A1 and B2, or linear peptides A2 and B1; Wherein, the cleavage reagent 1 is a mixed solution composed of one selected from the following reagents and a pyridyl disulfide compound: TFA / Tis / H2O / m-cresol, TFA / Tis / H2O, TFA / Tis / Phenol / H2O / m-cresol, TFA / Tis / Phenol / m-cresol; the pyridyl disulfide compound is selected from 2,2'-dithiobis(5-nitropyridine), dithiooxypyridine, 2,2'-dithiodipyridine, and 4,4'-dithiodipyridine; The cleavage reagent 2 is selected from one of TFA / Tis / H2O / m-cresol, TFA / Tis / H2O, TFA / Tis / Phenol / m-cresol, TFA / Mpr / H2O / m-cresol, TFA / Tis / Mpr / m-cresol, TFA / Tis / Phenol / Mpr / m-cresol; Wherein, the acidic solution is selected from acetic acid, hydrochloric acid, phosphoric acid, or their buffer salt solutions; Wherein, the second cyclization adopts the iodine oxidation method or the hydrogen peroxide oxidation method.

2. The method for the directed synthesis of the polypeptide dimer according to claim 1, wherein The pyridyl disulfide compound described is 2,2'-dithiopyridine. The synthesis of the cis-polypeptide dimer is specifically carried out in the following steps: (1) Prepare a linear peptide resin A containing aa1(Trt) and aa2(Acm); (2) Cleave resin A with cleavage reagent 1 to obtain a linear peptide A1 containing aa1(S-Pyr) and aa2(Acm); cleave resin A with cleavage reagent 2 to obtain a linear peptide A2 containing aa1 and aa2(Acm); (3) Take linear peptides A1 and A2 and perform the first cyclization in an acidic solution; (4) Remove Acm and perform the second cyclization to obtain the cis-polypeptide dimer; Or (1) Prepare a linear peptide resin B containing aa1(Acm) and aa2(Trt); (2) Cleave resin B with cleavage reagent 1 to obtain a linear peptide B1 containing aa1(Acm) and aa2(S-Pyr); cleave resin B with cleavage reagent 2 to obtain a linear peptide B2 containing aa1(Acm) and aa2; (3) Take linear peptides B1 and B2 and perform the first cyclization in an acidic solution; (4) Remove Acm and perform the second cyclization to obtain the cis-polypeptide dimer.

3. The method for the directed synthesis of the polypeptide dimer according to claim 1, wherein The pyridyl disulfide compound described is 2,2'-dithiopyridine. The synthesis of the trans-polypeptide dimer is specifically carried out in the following steps: (1) Prepare a linear peptide resin A containing aa1(Trt) and aa2(Acm), and a linear peptide resin B containing aa1(Acm) and aa2(Trt); (2) Cleave resins A and B with cleavage reagent 1 to obtain a linear peptide A1 containing aa1(S-Pyr) and aa2(Acm), and a linear peptide B1 containing aa1(Acm) and aa2(S-Pyr); cleave resins A and B with cleavage reagent 2 to obtain a linear peptide A2 containing aa1 and aa2(Acm), and a linear peptide B2 containing aa1(Acm) and aa2; (3) Take linear peptides A1 and B2 and perform the first cyclization in an acidic solution; (4) Remove Acm and perform the second cyclization to obtain the trans-polypeptide dimer; Or (3) Take linear peptides A2 and B1 and perform the first cyclization in an acidic solution; (4) Remove Acm and perform the second cyclization to obtain the trans-polypeptide dimer.

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