Atosiban and methods for inhibiting the formation of its polymeric impurities

Abstract

This invention relates to atosiban and a method for inhibiting the formation of polymeric impurities thereof. The method involves sequentially coupling amino acids with N-terminal Fmoc protection and side-chain protection according to the main peptide sequence of atosiban, comprising the following steps: using Fmoc-rink Amide AM resin as a starting material, performing a peptide linking reaction using a mixture of HOBT and DIC as a condensing agent, sequentially linking eight Fmoc-protected amino acids and Mpr(Trt)-OH to obtain a linear atosiban resin; adding iodine to the linear atosiban resin for oxidation, causing a disulfide bond to form between the 1-position MPa and the 6-position Cys to obtain a cyclized atosiban resin; adding a cleavage reagent to the obtained cyclized atosiban resin for cleavage, and separating to obtain atosiban. This invention also relates to atosiban prepared by the aforementioned method and their use in the preparation of oxytocin competitive antagonists for use in pregnant women in need to delay impending preterm labor.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a synthetic cyclic polypeptide composed of 9 amino acids. In particular, it relates to an oxytocin competitive antagonist for use in pregnant women in need to delay impending preterm labor. It also relates to atosiban or a pharmaceutical salt thereof, such as acetate, and a method for synthesizing the cyclic polypeptide, especially a method that can inhibit the formation of polymers, especially dimer impurities, in order to obtain atosiban of excellent chemical purity. Background Technology

[0002] Atosiban acetate injection was originally developed by Ferring AB and was first licensed for marketing by EMEA in 2000. Its trade name is [Brand Name Missing]. It was approved for marketing in China in 2006. Currently, the indications for atosiban in clinical practice at home and abroad are: to delay impending preterm labor in pregnant women with the following conditions: regular uterine contractions lasting at least 30 seconds each, ≥4 times every 30 minutes; cervical dilation of 1-3 cm (0-3 cm for nulliparous women) and uterine softening / thinning ≥50%; age ≥18; gestational age 24 to 33 weeks; and normal fetal heart rate.

[0003] Atosiban is an oxytocin analogue, a synthetic cyclic polypeptide that exists in the form of acetate in pharmaceuticals. It is a competitive antagonist of oxytocin receptors in the uterus and fetal membranes, and is the only uterus-specific uterine contraction inhibitor used to treat preterm labor. Its mechanism of action is to compete with oxytocin for oxytocin receptors in the myometrium and fetal membranes, reducing the efficacy of oxytocin and decreasing the calcium ion level on myocytes, thereby inhibiting uterine contractions.

[0004] The Chinese chemical name of atosiban is 1-(3-Mercaptopropanoic acid)-2-(O-ethyl-D-tyrosine)-4-L-threonine-8-L-ornithineoxytocin, and its English chemical name is 1-(3-Mercaptopropanoic acid)-2-(O-ethyl-D-tyrosine)-4-L-threonine-8-L-ornithineoxytocin. Its CAS registry number is 90779-69-4, its molecular formula is C43H67N11O12S2, its molecular weight is 994.19, and its chemical structural formula is:

[0005]

[0006] Atosiban is also represented in this field using the following peptide chain form:

[0007] Alternatively, it can be represented in the following peptide chain form:

[0008] c[Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys]-Pro-Orn-Gly-NH2,

[0009] The final product, atosiban, is formed by the cyclization of 3-thiol propanol acid with the thiol group on cysteine ​​to form a cyclic nonapeptide. After obtaining the uncyclized linear nonapeptide resin by solid-phase synthesis, a cyclization treatment is required to cyclize the two thiol groups of the linear nonapeptide resin.

[0010] Numerous methods for preparing atosiban acetate have been reported in the literature. For example, CN101696236B (application number 200910008880.6) discloses a solid-phase synthesis method for atosiban, comprising the following steps: 1) using Rink Amide resin as a carrier, deprotecting it with Fmoc- to obtain H2N-Rink Amide resin; 2) using HOBT and DIPCI as condensation reagents to link the carboxyl group of Fmoc-Gly-OH to the amino group of the resin to obtain Fmoc-Gly (resin); 3) sequentially synthesizing the remaining amino acids in the sequence using the Fmoc strategy in a solid phase; 4) performing solid-phase cyclization with iodine; 5) then cleaving with a cleavage reagent (trifluoroacetic acid / thioanisole / 1,2-ethylenedithiol / water), followed by ether precipitation to obtain crude atosiban peptide; 6) the crude product is purified by HPLC to obtain pure atosiban.

[0011] CN101314613B(200810043347.9) relates to a synthetic process for atosiban. The synthetic process steps are as follows: using aminomethyl resin as a carrier, amino acids are linked one by one according to the Fmoc / tbu solid-phase peptide synthesis method, and then sulfur-sulfur bonds are formed by iodine oxidation on the resin. Finally, the peptide is cleaved from the resin using a mixed solution of trifluoroacetic acid:triisopropylsilane:water:p-cresol to directly obtain the crude product with sulfur-sulfur bonds. Atosiban is obtained by preparation, HPLC purification and freezing.

[0012] CN102146121A (201010552213.7) relates to a method for solid-phase synthesis of atosiban, comprising the following steps: 1) using an amino resin as a carrier for deprotection; 2) linking the carboxyl group of Fmoc-Gly-OH to the amino group of the resin to obtain Fmoc-Gly-amino resin; 3) sequentially synthesizing the remaining protected amino acids in the solid phase; 4) removing the side chain protecting groups of cysteine ​​and mercaptopropionic acid; 5) performing solid-phase cyclization with iodine; 6) cleavage to obtain crude atosiban peptide; 7) purification to obtain pure atosiban.

[0013] CN111393508A (application number 202010226143.X) relates to a method for preparing atosiban. The method includes using different combinations of condensing agents / activators for different amino acids and different sites: DIC / HOBT, TBTU, TBTU / HOBT, HBTU / HOBT, BOP / HOBT, to prepare Mpr(Trt)-D-Tyr(OEt)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin, followed by peptide cleavage, oxidation, and purification to obtain atosiban.

[0014] CN114685614A (application number 202011609835.9) discloses a solid-phase synthesis method for atosiban, comprising the following steps: using Sieber Resin as the starting resin to synthesize Fmoc-Gly-Sieber Resin; sequentially coupling the corresponding protected amino acids or fragments according to the solid-phase synthesis method to obtain atosiban linear peptide resin, wherein the 8th amino acid is Fmoc-Orn(Dde)-OH; removing the Dde protection of Fmoc-Orn(Dde)-OH with hydrazine hydrate, followed by direct solid-phase oxidation, and cleavage to obtain atosiban.

[0015] CN115461355A (application number 202080099727.3) provides a method for preparing atosiban, comprising the following steps: (a) using Fmoc-Gly-Rink resin as a carrier, coupling amino acids protected by Fmoc at the N-terminus one by one to the Fmoc-Gly-Rink resin in the presence of a peptide coupling agent and performing Fmoc deprotection one by one, thereby preparing a linear crude atosiban peptide in the solid phase; (b) dissolving the linear crude atosiban peptide obtained in step (a) using a dissolving solution to obtain a linear crude atosiban peptide solution, and using a buffer solution and an oxidizing agent solution to form a disulfide bond between MPa at position 1 and Cys at position 6 of the linear crude atosiban peptide solution, thereby preparing a cyclized crude atosiban peptide in the liquid phase; and (c) purifying the cyclized crude atosiban peptide obtained in step (b) to obtain purified atosiban.

[0016] CN115038711A (application number 202180001434.1) discloses a method for synthesizing atosiban, comprising the following steps: synthesizing Fmoc-Pro-Orn-Gly-NH2 tripeptide, using triphenylmethyl resin as the starting resin to obtain Fmoc-Pro-Orn (triphenylmethyl resin)-Gly-NH2; sequentially inserting the corresponding protected amino acids or fragments into the atosiban sequence using a condensing agent to obtain atosiban linear peptide resin; and obtaining atosiban through cleavage and oxidation.

[0017] After obtaining the cyclic atosiban nonapeptide, purification is required, typically using preparative liquid chromatography (HPLC). It has been found that dimer impurities are easily generated during the cyclization of linear atosiban peptide resin to form the cyclic atosiban nonapeptide resin. The formation of these dimer impurities inevitably increases the burden on subsequent purification, especially when using preparative HPLC. Therefore, reducing the formation of dimer impurities during cyclization is of significant practical importance for the preparation of atosiban. Summary of the Invention

[0018] The object of this invention is to provide a novel method for preparing atosiban, such as its acetate, with the aim of achieving one or more technical advantages, such as minimizing the generation of dimer impurities during cyclization, thereby reducing the chromatographic burden of subsequent purification. It has been unexpectedly discovered that the method of this invention exhibits one or more superior technical advantages as described in this invention in the process of preparing atosiban or its acetate, and this invention is based on such discoveries.

[0019] Therefore, the first aspect of the present invention provides a method for preparing atosiban using a solid-phase synthesis, wherein amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled according to the main peptide sequence of atosiban, comprising the following steps:

[0020] (i) Using Fmoc-rink Amide AM resin as the starting material, a mixture of HOBT and DIC was used as a condensing agent to carry out the peptide linkage reaction, sequentially linking Fmoc-Gly-OH, Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, and Mpr(Trt)-OH. Before each peptide linkage reaction, the fluorene methoxycarbonyl group was removed sequentially using a decapping agent to obtain linear atosiban resin.

[0021] (ii) Iodine is added to linear atosiban resin to carry out an oxidation reaction, so that the first position MPa and the sixth position Cys form a disulfide bond to obtain cyclized atosiban resin.

[0022] (iii) Add a cleavage reagent to the obtained cyclized atosiban resin to perform cleavage, and separate atosiban; optionally,

[0023] (iv) The crude atosiban was purified by preparative liquid chromatography to obtain purified atosiban.

[0024] According to the method of the first aspect of the invention, the decapping agent used is 30% piperidine / DMF.

[0025] According to the method of the first aspect of the present invention, the amount of the decapping agent used is such that its feeding ratio with the resin is 4 to 6 ml / g resin, for example 5 to 5.5 ml / g resin.

[0026] According to the method of the first aspect of the present invention, the conditions for the decapping reaction are a reaction at 40-50°C for 20-30 minutes, for example, a decapping reaction at 45°C for 25 minutes.

[0027] According to the method of the first aspect of the present invention, the peptide-addition reagent used in the peptide-addition reaction is DMF.

[0028] According to the method of the first aspect of the invention, the amount of Fmoc-protected amino acids used in each peptide-linking reaction is 2 to 4 times the amount of resin, for example, 3 times the amount of resin. According to the method of the first aspect of the invention, the amount of amino acids used in the final peptide-linking reaction is 4 to 5 times the amount of resin, for example, 4 times the amount of resin.

[0029] According to the method of the first aspect of the present invention, in the peptide inoculation reaction, the molar amount of HOBT used is 2 to 5 times, for example, 3 to 4 times, that of the resin. For example, when adding the 1st to 8th amino acids, the molar amount of HOBT is 3 times that of the resin, and when adding the 9th amino acid, the molar amount of HOBT is 4 times that of the resin. The molar amount of DIC is 2 to 6 times, for example, 3 to 5 times, that of the resin. For example, when adding the 1st to 8th amino acids, the molar amount of DIC is 3.6 times that of the resin, and when adding the 9th amino acid, the molar amount of DIC is 4.8 times that of the resin.

[0030] According to the method of the first aspect of the present invention, the temperature of the peptide inoculation reaction is 25-35°C, for example 30°C, the reaction time is 24 hours when the first amino acid is inoculated, and the reaction time is 1-2 hours, for example 1.5 hours, when subsequent amino acids are inoculated.

[0031] According to the method of the first aspect of the invention, the molar amount of iodine used in the cyclization reaction is 5 to 10 times, for example, 8 times, the amount of resin; for example, the solvent used in the cyclization reaction is DMF, and the amount of solvent used per 1g of iodine is 20 to 50 ml, for example, 25 to 35 ml, for example, about 31 ml. In another embodiment, the solvent used in the cyclization reaction is acetone, and the amount of acetone used per 1g of iodine is 20 to 50 ml, for example, 25 to 35 ml, for example, about 31 ml. In another embodiment, the solvent used in the cyclization reaction is acetone and formic acid is also added, and the amount of acetone used per 1g of iodine is 20 to 50 ml, for example, 25 to 35 ml, for example, about 31 ml; the volume ratio of acetone to formic acid is 200:0.5 to 2, for example, 200:1. For example, the cyclization reaction is carried out under nitrogen stirring for 20-60 minutes, for example, 30 minutes; for example, after the cyclization reaction is completed, the mixture is washed sequentially with DMF, DCM and methanol, for example, three times with each solvent; for example, after washing, it is dried under vacuum. For example, the cyclization reaction is carried out as follows: based on 80 mmol resin feed, 162.6 g of iodine is weighed and added to a mixture of 5000 ml acetone and 25 ml formic acid and stirred to dissolve. After complete dissolution, it is added to the reaction vessel and stirred under nitrogen for 30 minutes; after the reaction is completed, the reaction solution is removed, and the mixture is washed three times with DMF at 3000 ml / time, then three times with DCM at 3000 ml / time, and finally three times with methanol at 3000 ml / time. The mixture is then dried under vacuum, poured out, and placed in a vacuum drying oven for 12 hours to obtain cyclized atosiban resin.

[0032] According to the method of the first aspect of the present invention, a mixture of trifluoroacetic acid, thioanisole, 1,2-ethanedithiol and water is used as a peptide cleavage reagent (cleavage reagent) to carry out the peptide cleavage reaction (cleavage reaction).

[0033] According to the method of the first aspect of the present invention, the peptide-cleaving reagent is a mixture of trifluoroacetic acid: thioanisole: 1,2-ethylenedithiol: water = 90:5:3:2.

[0034] According to the method of the first aspect of the present invention, the peptide cleavage reaction conditions are as follows: peptide cleavage reaction at 30-40°C for 1-3 hours, for example, peptide cleavage reaction at 32°C for 2 hours.

[0035] According to the method of the first aspect of the present invention, the precipitate obtained by the peptide cleavage reaction is precipitated and washed with diethyl ether, and then dried to obtain crude atosiban.

[0036] According to the method of the first aspect of the invention, a preparative C18 column is used for purification.

[0037] According to the method of the first aspect of the present invention, the mobile phase A used for purification with a preparative C18 column is a 0.3% aqueous acetic acid solution, and the mobile phase B is acetonitrile; the elution program is as follows: 0-10 min maintain mobile phase B at 5%, 10-11 min increase mobile phase B to 35%, 11-70 min linearly increase mobile phase B to 45%, 70-71 min increase mobile phase B to 50%, and 71-75 min maintain mobile phase B at 50%.

[0038] According to the method of the first aspect of the present invention, it is prepared by a method comprising the following steps:

[0039] (1) Preparation of Fmoc-Gly-resin: Add 80 mmol of Fmoc-rink Amide AM resin to the synthesis reactor, wash with DCM, then wash with DMF, add 550 ml of prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, wash with DMF, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Gly-OH and 240 mmol of HOBT, add N,N-dimethylformamide and stir to dissolve, after complete dissolution, add 290 ml of 1 mol / L DIC / DMF solution, stir evenly and add to the reactor, stir and react for 24 hours, after the reaction is complete, remove the reaction solution, wash with DMF 3 times, then wash with DCM, and finally wash with methanol, dry, pour out, and put into a vacuum drying oven to dry for 12 h to obtain Fmoc-Gly-resin;

[0040] (2) Preparation of Fmoc-Orn(Boc)-Gly-resin: Add the Fmoc-Gly-resin obtained in the previous step to the reaction vessel, wash with DCM, and then wash with DMF; add 550 ml of the prepared 30% piperidine / DMF solution to the reaction vessel, stir and react for 30 min, remove the reaction solution, wash with DMF, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Orn(Boc)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve, after complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reaction vessel, stir and react for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin;

[0041] (3) Preparation of Fmoc-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Pro-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve, after complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin;

[0042] (4) Preparation of Fmoc-Cys(Trt)-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Cys(Trt)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin;

[0043] (5) Preparation of Fmoc-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Asn(Trt)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin;

[0044] (6) Preparation of Fmoc-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Thr(tBu)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin;

[0045] (7) Preparation of Fmoc-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Ile-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin;

[0046] (8) Preparation of Fmoc-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-D-Tyr(Et)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin;

[0047] (9) Preparation of Mpr(Trt)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash, and the ninhydrin test should be positive; weigh 320 mmol of Mpr(Trt)-OH and 320 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve, after complete dissolution, add 385 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin, i.e., linear atosiban resin;

[0048] (10) Cyclization: Weigh 162.6g of iodine and add it to a mixture of 5000ml of acetone and 25ml of formic acid. Stir and dissolve the mixture. After it is completely dissolved, add it to the reaction vessel and stir with nitrogen for 30min. After the reaction is complete, remove the reaction solution, wash with DMF, then wash with DCM, and finally wash with methanol. Dry the solution, pour it out, and vacuum dry it to obtain cyclized atosiban resin.

[0049] (11) Cleavage: Add the cyclized resin obtained in the previous step to the reaction flask, add 2200 ml of the prepared cleavage reagent (trifluoroacetic acid: thioanisole: 1,2-ethylenedithiol: water = 90%: 5%: 3%: 2%), stir and react for 120 min; filter, wash the resin with a small amount of trifluoroacetic acid, filter again; combine the filtrates, slowly settle into anhydrous ether; let stand at 5±2℃ for 2 h to settle, then start centrifugation, wash with anhydrous ether after centrifugation, centrifuge again, and vacuum dry to obtain atosiban.

[0050] According to the method of the first aspect of the present invention, it further includes purifying the pyrolyzed atosiban by the following steps: dissolving the pyrolyzed atosiban in water, filtering with a microporous membrane, loading the filtrate onto a preparative high-performance liquid chromatograph according to purification conditions, collecting the target peak solution, concentrating the collected solution, desalting, and freeze-drying to obtain purified atosiban; the purification conditions are: octadecylsilane-bonded silica gel column, 10 μm. Detection wavelength: 230 nm; flow rate: 450 ml / min; mobile phase A: 0.3% acetic acid aqueous solution; mobile phase B: acetonitrile; elution program: 0–10 min maintain mobile phase B at 5%; 10–11 min increase mobile phase B to 35%; 11–70 min linearly increase mobile phase B to 45%; 70–71 min increase mobile phase B to 50%; 71–75 min maintain mobile phase B at 50%.

[0051] Furthermore, a second aspect of the present invention provides atosiban prepared by the method described in any one of the first aspects of the present invention.

[0052] Furthermore, a third aspect of the invention provides the use of atosiban prepared by the method of any one of the first aspects of the invention or atosiban of the second aspect in the preparation of an oxytocin competitive antagonist for use in pregnant women in need to delay impending preterm labor.

[0053] The atosiban prepared by the method of the present invention exhibits one or more excellent technical effects as described in the context herein. Attached Figure Description

[0054] Figure 1 This is a chromatogram of a typical system suitability test solution for related substance testing. Detailed Implementation

[0055] The present invention provides the following examples to further illustrate various aspects of the invention. Some materials and their abbreviations used herein have their general meanings in the art, for example:

[0056] N α -(9-fluorenylmethoxycarbonyl)-glycine, Fmoc-Gly-OH, Mr. 297.32;

[0057] N α -(9-fluorenylmethoxycarbonyl)-N δ -tert-Butyloxycarbonyl-L-ornithine, Fmoc-Orn(Boc)-OH, Mr. 454.52;

[0058] N α -(9-fluorenylmethoxycarbonyl)-L-proline, Fmoc-Pro-OH, Mr. 337.38;

[0059] N α -(9-fluorenylmethoxycarbonyl)-S-triphenylmethyl-L-cysteine, Fmoc-Cys(Trt)-OH, Mr. 585.72;

[0060] N α -(9-fluorenylmethoxycarbonyl)-N δ -Triphenylmethyl-L-asparagine, Fmoc-Asn(Trt)-OH, Mr. 596.70;

[0061] N α -(9-fluorenylmethoxycarbonyl)-O-tert-butyl-L-threonine, Fmoc-Thr(tBu)-OH, Mr. 397.50;

[0062] N α-(9-fluorenylmethoxycarbonyl)-L-isoleucine, Fmoc-Ile-OH, Mr. 353.40;

[0063] N α -(9-fluorenylmethoxycarbonyl)-O-ethyl-D-tyrosine, Fmoc-D-Tyr(Et)-OH, Mr. 431.48;

[0064] S-Triphenylmethyl-mercaptopropionic acid, Mpa(Trt)-OH, Mr. 348.46;

[0065] 1-Hydroxybenzotriazole, HOBT, Mr. 135.12;

[0066] N,N-Diisopropylcarbodiimide, DIC, Mr. 126.20.

[0067] All other unlisted abbreviations or markings have meanings well-known in the art, and these materials are readily available from commercial sources; for example, Fmoc-Gly-OH, Fmoc-Orn(Boc)-OH, and Fmoc-Pro-OH were all purchased from GLPBIO and their stated HPLC purities are all greater than 98%. All materials used in this document are from the same batch unless otherwise stated.

[0068] This invention employs a solid-phase synthesis method, using Fmoc-rink Amide AM resin as the starting resin, and sequentially coupling amino acids with N-terminal Fmoc protection and side chain protection according to the atosiban backbone peptide sequence. In specific examples of this invention, unless otherwise specified, the decapping reaction is carried out at 45°C for 30 min using a 30% piperidine / DMF solution as the decapping reagent; in specific examples of this invention, unless otherwise specified, the peptide incorporation reaction after the addition of HOBT and DIC is carried out at 30°C; in specific examples of this invention, unless otherwise specified, the cleavage / peptide release reaction is carried out at 32°C; these reaction conditions are conventional in the art.

[0069] Example 1: Preparation of atosiban

[0070] This embodiment refers to the method described in document CN101696236B of the inventors' research team for preparing atosiban.

[0071] (1) Preparation of Fmoc-Gly-resin

[0072] Add 80 mmol (105.4 g) of Fmoc-rink Amide AM resin to the synthesis reactor, wash three times with 550 ml of DCM each time, then wash three times with 550 ml of DMF each time, add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, wash six times with 550 ml of DMF each time, and test the deprotection effect with 5% ninhydrin ethanol solution (if the resin ninhydrin test is positive, the coupling of the first amino acid can be carried out).

[0073] Weigh 240 mmol of Fmoc-Gly-OH and 240 mmol of HOBT, add N,N-dimethylformamide (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of 1 mol / L DIC / DMF solution, stir evenly and add to the reaction vessel. Stir and react for 24 hours. After the reaction is complete (the coupling effect should be negative when tested with ninhydrin), remove the reaction solution, wash 3 times with DMF (550 ml each time), wash 3 times with DCM (550 ml each time), and finally wash 3 times with methanol (550 ml each time). Drain, pour out, and place in a vacuum drying oven to dry for 12 hours to obtain Fmoc-Gly-resin.

[0074] The present invention provides a method for detecting ninhydrin: Resin is taken from the reaction vessel using a stainless steel sampler and added to a detection tube for ninhydrin detection (2-4 drops of coupling solution are added to the detection tube, and 5-10 drops of DMF are added with a dropper to wash the sampled resin twice). A 5% ninhydrin / ethanol (M / V) solution is added, and the mixture is heated at 100℃-150℃ for 5 minutes using an electric furnace. If the resin turns blue or shows a significant color change, it indicates that the amino group of the resin is exposed, indicating a positive result; if the resin is colorless or pale yellow and transparent, it indicates that the amino group of the resin is protected, indicating a negative result. Preparation of the 5% ninhydrin / ethanol (M / V) solution: To prepare 100ml of 5% ninhydrin / ethanol (M / V) solution, first weigh 5g of ninhydrin, then measure 100ml of anhydrous ethanol and pour it into a beaker. Stir until dissolved evenly, then transfer to a brown bottle for later use.

[0075] (2) Preparation of Fmoc-Orn(Boc)-Gly-resin

[0076] Add the Fmoc-Gly-resin obtained in the previous step to the reactor, wash with DCM 3 times each time (550ml), and then wash with DMF 3 times each time (550ml).

[0077] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0078] Weigh 240 mmol of Fmoc-Orn(Boc)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0079] (3) Preparation of Fmoc-Pro-Orn(Boc)-Gly-resin:

[0080] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0081] Weigh 240 mmol of Fmoc-Pro-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0082] (4) Preparation of Fmoc-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0083] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0084] Weigh 240 mmol of Fmoc-Cys(Trt)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0085] (5) Preparation of Fmoc-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0086] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0087] Weigh 240 mmol of Fmoc-Asn(Trt)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0088] (6) Preparation of Fmoc-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0089] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0090] Weigh 240 mmol of Fmoc-Thr(tBu)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0091] (7) Preparation of Fmoc-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0092] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0093] Weigh 240 mmol of Fmoc-Ile-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0094] (8) Preparation of Fmoc-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0095] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0096] Weigh 240 mmol of Fmoc-D-Tyr(Et)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash with DMF 6 times (550 ml each time), and dry to obtain the title resin.

[0097] (9) Preparation of Mpr(Trt)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0098] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0099] Weigh 320 mmol of Mpr(Trt)-OH and 320 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 385 ml of DMF solution with 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash with DMF 6 times (550 ml each time), and dry to obtain the title resin, i.e., linear atosiban resin.

[0100] (10) Cycloning

[0101] Weigh 162.6g of iodine and add it to 5000ml of DMF and stir to dissolve. After complete dissolution, add it to the reaction vessel and stir with nitrogen for 30min. After the reaction is complete, remove the reaction solution, wash 3 times with DMF at 3000ml / time, wash 3 times with DCM at 3000ml / time, and finally wash 3 times with methanol at 3000ml / time. Dry the solution, pour it out, and place it in a vacuum drying oven to dry for 12h to obtain cyclized atosiban resin.

[0102] (11) Pyrolysis

[0103] Add the cyclized resin obtained in the previous step to a round-bottom flask, add 2200 ml of the prepared lysis reagent (trifluoroacetic acid: thioanisole: 1,2-ethanedithiol: water = 90%: 5%: 3%: 2%), stir and react for 120 min; filter, wash the resin three times with a small amount of trifluoroacetic acid (the amount of detergent just enough to cover the resin), filter again; combine the filtrates, slowly settle into 22000 ml of anhydrous diethyl ether; let stand at 5±2℃ for 2 h to settle, then start centrifugation, after centrifugation, wash 6 times with 2500 ml of anhydrous diethyl ether each time and centrifuge again, put the obtained crude peptide into a vacuum drying oven to dry, thus obtaining the crude oxidized atosiban.

[0104] (12) Purification: The crude peptide obtained by vacuum drying was dissolved in water, and the solution was filtered through a 0.45 μm microporous membrane. The filtrate was loaded onto the sample according to the purification conditions, the target peak solution was collected, and the collected solution was concentrated, desalted, and freeze-dried to obtain the target product atosiban, with a yield >65%. The purification conditions were as follows:

[0105] Instrument: DAC150-LC6000 preparative high-performance liquid chromatograph;

[0106] Column: Octadecylsilane-bonded silica gel, 10 μm.

[0107] Sample loading amount: ≤50g target peptide / time;

[0108] Detection wavelength: 230nm;

[0109] Flow rate: 450 ml / min;

[0110] Mobile phase A is a 0.3% aqueous acetic acid solution, and mobile phase B is acetonitrile;

[0111] Elution program: 0–10 min maintain mobile phase B at 5%, 10–11 min increase mobile phase B to 35%, 11–70 min linearly increase mobile phase B to 45%, 70–71 min increase mobile phase B to 50%, 71–75 min maintain mobile phase B at 50%.

[0112] Example 2: Preparation of atosiban

[0113] (1) Preparation of Fmoc-Gly-resin

[0114] Add 80 mmol (calculated based on the degree of substitution) of Fmoc-rink Amide AM resin to the synthesis reactor. Wash three times with DCM (550 ml each time), then wash three times with DMF (550 ml each time). Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor and stir for 30 min. Remove the reaction solution and wash six times with DMF (550 ml each time). Detect the deprotection effect with 5% ninhydrin ethanol solution (if the resin ninhydrin test is positive, the coupling of the first amino acid can proceed).

[0115] Weigh 240 mmol of Fmoc-Gly-OH and 240 mmol of HOBT, add N,N-dimethylformamide (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of 1 mol / L DIC / DMF solution, stir evenly and add to the reaction vessel. Stir and react for 24 hours. After the reaction is complete (the coupling effect should be negative when tested with ninhydrin), remove the reaction solution, wash 3 times with DMF (550 ml each time), wash 3 times with DCM (550 ml each time), and finally wash 3 times with methanol (550 ml each time). Drain, pour out, and place in a vacuum drying oven to dry for 12 hours to obtain Fmoc-Gly-resin.

[0116] (2) Preparation of Fmoc-Orn(Boc)-Gly-resin

[0117] Add the Fmoc-Gly-resin obtained in the previous step to the reactor, wash with DCM 3 times each time (550ml), and then wash with DMF 3 times each time (550ml).

[0118] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0119] Weigh 240 mmol of Fmoc-Orn(Boc)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0120] (3) Preparation of Fmoc-Pro-Orn(Boc)-Gly-resin:

[0121] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0122] Weigh 240 mmol of Fmoc-Pro-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0123] (4) Preparation of Fmoc-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0124] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0125] Weigh 240 mmol of Fmoc-Cys(Trt)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0126] (5) Preparation of Fmoc-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0127] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0128] Weigh 240 mmol of Fmoc-Asn(Trt)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0129] (6) Preparation of Fmoc-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0130] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0131] Weigh 240 mmol of Fmoc-Thr(tBu)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0132] (7) Preparation of Fmoc-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0133] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0134] Weigh 240 mmol of Fmoc-Ile-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0135] (8) Preparation of Fmoc-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0136] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0137] Weigh 240 mmol of Fmoc-D-Tyr(Et)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash with DMF 6 times (550 ml each time), and dry to obtain the title resin.

[0138] (9) Preparation of Mpr(Trt)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0139] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0140] Weigh 320 mmol of Mpr(Trt)-OH and 320 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 385 ml of DMF solution with 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash with DMF 6 times (550 ml each time), and dry to obtain the title resin, i.e., linear atosiban resin.

[0141] (10) Cycloning

[0142] Weigh 162.6g of iodine and add it to a mixture of 5000ml of acetone and 25ml of formic acid. Stir and dissolve the iodine. After complete dissolution, add the solution to a reaction vessel and stir under nitrogen for 30 minutes. After the reaction is complete, remove the reaction solution and wash it three times with DMF at 3000ml each time, then three times with DCM at 3000ml each time, and finally three times with methanol at 3000ml each time. Dry the solution, pour it out, and place it in a vacuum drying oven to dry for 12 hours to obtain cyclized atosiban resin.

[0143] (11) Pyrolysis

[0144] Add the cyclized resin obtained in the previous step to a round-bottom flask, add 2200 ml of the prepared lysis reagent (trifluoroacetic acid: thioanisole: 1,2-ethanedithiol: water = 90%: 5%: 3%: 2%), stir and react for 120 min; filter, wash the resin three times with a small amount of trifluoroacetic acid (the amount of detergent just enough to cover the resin), filter again; combine the filtrates, slowly settle into 22000 ml of anhydrous diethyl ether; let stand at 5±2℃ for 2 h to settle, then start centrifugation, after centrifugation, wash 6 times with 2500 ml of anhydrous diethyl ether each time and centrifuge again, put the obtained crude peptide into a vacuum drying oven to dry, thus obtaining the crude oxidized atosiban.

[0145] Alternatively, the above-mentioned crude oxidized atosiban can be further purified by referring to the method in step (12) of Example 1 to obtain the target product atosiban.

[0146] Example 3: Preparation of atosiban

[0147] (1) Preparation of Fmoc-Gly-resin

[0148] Add 80 mmol (calculated based on the degree of substitution) of Fmoc-rink Amide AM resin to the synthesis reactor. Wash three times with DCM (550 ml each time), then wash three times with DMF (550 ml each time). Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor and stir for 30 min. Remove the reaction solution and wash six times with DMF (550 ml each time). Detect the deprotection effect with 5% ninhydrin ethanol solution (if the resin ninhydrin test is positive, the coupling of the first amino acid can proceed).

[0149] Weigh 240 mmol of Fmoc-Gly-OH and 240 mmol of HOBT, add N,N-dimethylformamide (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of 1 mol / L DIC / DMF solution, stir evenly and add to the reaction vessel. Stir and react for 24 hours. After the reaction is complete (the coupling effect should be negative when tested with ninhydrin), remove the reaction solution, wash 3 times with DMF (550 ml each time), wash 3 times with DCM (550 ml each time), and finally wash 3 times with methanol (550 ml each time). Drain, pour out, and place in a vacuum drying oven to dry for 12 hours to obtain Fmoc-Gly-resin.

[0150] (2) Preparation of Fmoc-Orn(Boc)-Gly-resin

[0151] Add the Fmoc-Gly-resin obtained in the previous step to the reactor, wash with DCM 3 times each time (550ml), and then wash with DMF 3 times each time (550ml).

[0152] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0153] Weigh 240 mmol of Fmoc-Orn(Boc)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0154] (3) Preparation of Fmoc-Pro-Orn(Boc)-Gly-resin:

[0155] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0156] Weigh 240 mmol of Fmoc-Pro-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0157] (4) Preparation of Fmoc-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0158] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0159] Weigh 240 mmol of Fmoc-Cys(Trt)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0160] (5) Preparation of Fmoc-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0161] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0162] Weigh 240 mmol of Fmoc-Asn(Trt)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0163] (6) Preparation of Fmoc-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0164] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0165] Weigh 240 mmol of Fmoc-Thr(tBu)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0166] (7) Preparation of Fmoc-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0167] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0168] Weigh 240 mmol of Fmoc-Ile-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0169] (8) Preparation of Fmoc-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0170] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0171] Weigh 240 mmol of Fmoc-D-Tyr(Et)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash with DMF 6 times (550 ml each time), and dry to obtain the title resin.

[0172] (9) Preparation of Mpr(Trt)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0173] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0174] Weigh 320 mmol of Mpr(Trt)-OH and 320 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 385 ml of DMF solution with 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash with DMF 6 times (550 ml each time), and dry to obtain the title resin, i.e., linear atosiban resin.

[0175] (10) Cycloning

[0176] Weigh 162.6g of iodine and add it to 5000ml of acetone. Stir and dissolve the iodine. After it is completely dissolved, add it to the reaction vessel and stir with nitrogen for 30 minutes. After the reaction is complete, remove the reaction solution and wash it three times with DMF at 3000ml each time, then wash it three times with DCM at 3000ml each time, and finally wash it three times with methanol at 3000ml each time. Dry the solution, pour it out, and place it in a vacuum drying oven to dry for 12 hours to obtain cyclized atosiban resin.

[0177] (11) Pyrolysis

[0178] Add the cyclized resin obtained in the previous step to a round-bottom flask, add 2200 ml of the prepared lysis reagent (trifluoroacetic acid: thioanisole: 1,2-ethanedithiol: water = 90%: 5%: 3%: 2%), stir and react for 120 min; filter, wash the resin three times with a small amount of trifluoroacetic acid (the amount of detergent just enough to cover the resin), filter again; combine the filtrates, slowly settle into 22000 ml of anhydrous diethyl ether; let stand at 5±2℃ for 2 h to settle, then start centrifugation, after centrifugation, wash 6 times with 2500 ml of anhydrous diethyl ether each time and centrifuge again, put the obtained crude peptide into a vacuum drying oven to dry, thus obtaining the crude oxidized atosiban.

[0179] Alternatively, the above-mentioned crude oxidized atosiban can be further purified by referring to the method in step (12) of Example 1 to obtain the target product atosiban.

[0180] Example 4: Preparation of atosiban

[0181] (1) Preparation of Fmoc-Gly-resin

[0182] Add 80 mmol (calculated based on the degree of substitution) of Fmoc-rink Amide AM resin to the synthesis reactor. Wash three times with DCM (550 ml each time), then wash three times with DMF (550 ml each time). Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor and stir for 30 min. Remove the reaction solution and wash six times with DMF (550 ml each time). Detect the deprotection effect with 5% ninhydrin ethanol solution (if the resin ninhydrin test is positive, the coupling of the first amino acid can proceed).

[0183] Weigh 240 mmol of Fmoc-Gly-OH and 240 mmol of HOBT, add N,N-dimethylformamide (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of 1 mol / L DIC / DMF solution, stir evenly and add to the reaction vessel. Stir and react for 24 hours. After the reaction is complete (the coupling effect should be negative when tested with ninhydrin), remove the reaction solution, wash 3 times with DMF (550 ml each time), wash 3 times with DCM (550 ml each time), and finally wash 3 times with methanol (550 ml each time). Drain, pour out, and place in a vacuum drying oven to dry for 12 hours to obtain Fmoc-Gly-resin.

[0184] (2) Preparation of Fmoc-Orn(Boc)-Gly-resin

[0185] Add the Fmoc-Gly-resin obtained in the previous step to the reactor, wash with DCM 3 times each time (550ml), and then wash with DMF 3 times each time (550ml).

[0186] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0187] Weigh 240 mmol of Fmoc-Orn(Boc)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0188] (3) Preparation of Fmoc-Pro-Orn(Boc)-Gly-resin:

[0189] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0190] Weigh 240 mmol of Fmoc-Pro-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0191] (4) Preparation of Fmoc-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0192] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0193] Weigh 240 mmol of Fmoc-Cys(Trt)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0194] (5) Preparation of Fmoc-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0195] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0196] Weigh 240 mmol of Fmoc-Asn(Trt)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0197] (6) Preparation of Fmoc-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0198] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0199] Weigh 240 mmol of Fmoc-Thr(tBu)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0200] (7) Preparation of Fmoc-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0201] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0202] Weigh 240 mmol of Fmoc-Ile-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash 6 times with 550 ml of DMF each time, and dry to obtain the title resin.

[0203] (8) Preparation of Fmoc-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0204] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0205] Weigh 240 mmol of Fmoc-D-Tyr(Et)-OH and 240 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 290 ml of DMF solution of 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash with DMF 6 times (550 ml each time), and dry to obtain the title resin.

[0206] (9) Preparation of Mpr(Trt)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin:

[0207] Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash 6 times each time (550 ml), and test the deprotection effect with 5% ninhydrin ethanol solution (the resin ninhydrin test should be positive).

[0208] Weigh 320 mmol of Mpr(Trt)-OH and 320 mmol of HOBT, add DMF (400-500 ml) and stir to dissolve. After complete dissolution, add 385 ml of DMF solution with 1 mol / L DIC and stir evenly. Add the prepared amino acid coupling solution to the reaction vessel and stir to react for 90 min (until the ninhydrin test is negative). Remove the reaction solution, wash with DMF 6 times (550 ml each time), and dry to obtain the title resin, i.e., linear atosiban resin.

[0209] (10) Cycloning

[0210] Weigh 162.6g of iodine and add it to a mixture of 5000ml of DMF and 25ml of formic acid. Stir and dissolve the solution. After complete dissolution, add the solution to a reaction vessel and stir under nitrogen for 30 minutes. After the reaction is complete, remove the reaction solution and wash the solution three times with 3000ml of DMF each time, then three times with 3000ml of DCM each time, and finally three times with 3000ml of methanol each time. Dry the solution, pour it out, and place it in a vacuum drying oven to dry for 12 hours to obtain cyclized atosiban resin.

[0211] (11) Pyrolysis

[0212] Add the cyclized resin obtained in the previous step to a round-bottom flask, add 2200 ml of the prepared lysis reagent (trifluoroacetic acid: thioanisole: 1,2-ethanedithiol: water = 90%: 5%: 3%: 2%), stir and react for 120 min; filter, wash the resin three times with a small amount of trifluoroacetic acid (the amount of detergent just enough to cover the resin), filter again; combine the filtrates, slowly settle into 22000 ml of anhydrous diethyl ether; let stand at 5±2℃ for 2 h to settle, then start centrifugation, after centrifugation, wash 6 times with 2500 ml of anhydrous diethyl ether each time and centrifuge again, put the obtained crude peptide into a vacuum drying oven to dry, thus obtaining the crude oxidized atosiban.

[0213] Alternatively, the above-mentioned crude oxidized atosiban can be further purified by referring to the method in step (12) of Example 1 to obtain the target product atosiban.

[0214] Example 5: Atosiban was prepared according to the method of Example 2 of the present invention, except that formic acid was replaced with an equimolar amount of acetic acid during the cyclization process, and crude oxidized atosiban and purified atosiban were obtained.

[0215] Experimental Example 1: Determination of Related Substances

[0216] The following HPLC method was used to determine the relevant substances in the crude oxidized atosiban obtained in Examples 1-5 above:

[0217] 1) Test solution: Take an appropriate amount of sample and add mobile phase to prepare a solution containing about 1.5 mg per 1 ml, which is used as the test solution;

[0218] 2) Control solution: Measure 1 ml of the test solution, place it in a 100 ml volumetric flask, add mobile phase A to dilute to the mark, shake well, and use it as the control solution (1%).

[0219] 3) System suitability solution: Take 15 mg each of impurity 9 reference standard and impurity 27 reference standard, place them in the same 100 ml volumetric flask, add mobile phase A to dissolve and dilute to the mark, shake well, and prepare the impurity mixed stock solution; then take about 25 mg of atosiban acetate reference standard, place it in a 50 ml volumetric flask, add 1 ml of the impurity mixed stock solution, dilute to the mark with mobile phase A, shake well, and prepare a mixed solution containing about 3 μg each of impurity 9 and impurity 27 and 0.5 mg of atosiban acetate per ml;

[0220] 4) Inject 20 μl of the control solution into the liquid chromatograph, adjust the detection sensitivity so that the peak height of the main component is about 20% of the full scale, then accurately measure 20 μl each of the test solution and the control solution, inject them into the liquid chromatograph, record the chromatograms until twice the retention time of the main component peak, and calculate the content of related substances.

[0221] Chromatographic conditions: A ZORBAX column packed with octadecylsilane-bonded silica gel was used. Extend-C18 column (4.6 x 250 mm, 3.5 μm); using 0.05 mol / L phosphate buffer (prepared by dissolving 6.8 g potassium dihydrogen phosphate in 1000 ml of water, adjusting the pH to 2.3 with phosphoric acid, and then adding 120 μl of triethylamine) as mobile phase A and acetonitrile as mobile phase B for gradient elution; detection wavelength: 220 nm; injection volume: 30 μl; flow rate: 1.0 ml / min. The theoretical plate number, calculated based on the atosiban peak, should be no less than 5000; the gradient elution program is as follows:

[0222] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 28 73 27 44 60 40 49 60 40 50 80 20 60 80 20

[0223] System suitability requirements: In the chromatogram of the system suitability test solution, atosiban, impurity 27, and impurity 9 should elute sequentially. The resolution between the peaks of impurity 27 and impurity 9 should be greater than 1.2, and the theoretical plate number calculated based on the atosiban peak should be no less than 3000. A typical system suitability test solution chromatogram is shown below. Figure 1 As shown;

[0224] Determination method: Accurately measure the test solution and the control solution, inject them into the liquid chromatograph, record the chromatograms, and compare the peak areas of impurity 9 and impurity 27 with the main peak area of ​​the control solution to calculate their percentage content.

[0225] Results: The crude atosiban obtained from step (11) of Examples 1-5 was determined using the method of Example 1. The purity of the five products was calculated using the area normalization method, and the purity was 69.4%, 78.3%, 65.3%, 70.7%, and 71.2%, respectively. The difference in purity among the five samples was mainly due to the different amounts of impurity 9 and impurity 27. The percentage content of impurity 9 in the crude atosiban obtained in Examples 1-5 was 4.36%, 0.96%, 5.13%, 4.62%, and 3.93%, respectively, while the percentage content of impurity 27 in the crude atosiban obtained in Examples 1-4 was 4.74%, 1.13%, 5.92%, 4.47%, and 4.07%, respectively. The results above unexpectedly show that the content of the two dimer impurities in the crude atosiban obtained in Example 2 was significantly lower than that in the products obtained in the other examples. This indicates that the subsequent purification of the crude product can significantly reduce the loading of the preparative chromatographic column, reduce production costs, and improve production efficiency. In addition, the atosiban obtained by purification in steps (12) of Examples 1 to 5 was determined using the method of this Experimental Example 1. The purity of the five products was 93.2%, 96.6%, 91.6%, 92.8%, and 92.3%, respectively, calculated using the area normalization method. The impurity percentages of the five products were 0.94%, 0.23%, 0.79%, 0.84%, and 0.73%, respectively, and the impurity percentages of the five products were 1.13%, 0.31%, 0.95%, 1.03%, and 0.89%, respectively. The atosiban can be further purified in subsequent purification, salt conversion, and other processes.

[0226] In this invention, impurity 9 is: (3-mercaptopropionyl)-D-(O-ethyl)-tyrosyl-L-isoleucyl-L-threonyl-L-asparaginyl-L-cysteyl-L-prolyl-L-guanyl-glycineamide (1→1-disulfide bond, 6→6-disulfide bond), C 86 H 134 N 22 O 24 S4, 1988.38, the structural formula is:

[0227]

[0228] Impurity 27 is: (3-mercaptopropionyl)-D-(O-ethyl)-tyrosyl-L-isoleucyl-L-threonyl-L-asparaginyl-L-cysteyl-L-prolyl-L-guanyl-glycineamide (1→6-disulfide bond, 6→1-disulfide bond), C 86 H 134 N 22 O 24 S4, 1988.38, the structural formula is:

[0229]

[0230] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for preparing atosiban, comprising the sequential coupling of amino acids with N-terminal Fmoc protection and side chain protection according to the main peptide sequence of atosiban, including the following steps: (i) Using Fmoc-rink Amide AM resin as the starting material, a peptide linkage reaction was carried out using a mixture of HOBT and DIC as the condensing agent, sequentially linking Fmoc-Gly-OH, Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, and Mpr(Trt)-OH. Before each peptide linkage reaction, a decapping agent was used to remove the fluorene methoxycarbonyl group to obtain linear atosiban resin; the decapping agent was 30% piperazine. The dosage of DMF / DMC decapping reagent is as follows: its ratio to resin is 4-6 ml / g resin; the peptide inoculation reagent used in the peptide inoculation reaction is DMF; the amount of Fmoc-protected amino acid used in each peptide inoculation reaction except the last one is 2-4 times the amount of resin; the amount of Fmoc-protected amino acid used in the last peptide inoculation reaction is 4-5 times the amount of resin; the molar number of HOBT used in the peptide inoculation reaction is 2-5 times that of resin; the temperature of the peptide inoculation reaction is 25-35℃; the molar number of DIC is 3.6 times that of resin when inoculating the 1st to 8th amino acids, and the molar number of DIC is 4.8 times that of resin when inoculating the 9th amino acid; (ii) Iodine was added to linear atosiban resin to carry out an oxidation reaction, so that disulfide bonds were formed between the 1st position MPa and the 6th position Cys to obtain cyclized atosiban resin. The operation was as follows: 162.6 g of iodine was weighed based on 80 mmol of resin feed and added to a mixture of 5000 ml acetone and 25 ml formic acid. After complete dissolution, the solution was added to a reaction vessel and stirred under nitrogen for 30 min. After the reaction was completed, the reaction solution was removed, and the solution was washed 3 times with DMF at 3000 ml / time, then washed 3 times with DCM at 3000 ml / time, and finally washed 3 times with methanol at 3000 ml / time. The solution was then dried under vacuum, poured out, and placed in a vacuum drying oven for 12 h to obtain cyclized atosiban resin. (iii) Add a pyrolysis reagent to the obtained cyclized atosiban resin to pyrolyze it and separate crude atosiban; the pyrolysis reagent is a mixture of trifluoroacetic acid: thioanisole: 1,2-ethylenedithiol: water = 90:5:3:2; the pyrolysis reaction conditions are pyrolysis at 30~40℃ for 1~3 hours; the precipitate obtained by the pyrolysis reaction is precipitated and washed with diethyl ether, and dried to obtain crude atosiban; (iv) The crude atosiban was purified by preparative C18 column liquid chromatography to obtain purified atosiban. The mobile phase A used for purification was 0.3% aqueous acetic acid solution, and the mobile phase B was acetonitrile. The elution program was as follows: 0-10 min maintain mobile phase B at 5%, 10-11 min increase mobile phase B to 35%, 11-70 min linearly increase mobile phase B to 45%, 70-71 min increase mobile phase B to 50%, and 71-75 min maintain mobile phase B at 50%.

2. The method according to claim 1, wherein the amount of the decapping agent is: its feeding ratio with the resin is 5~5.5 ml / g resin.

3. The method according to claim 1, wherein the decapping reaction is carried out at 40-50°C for 20-30 minutes.

4. The method according to claim 1, wherein the decapping reaction is carried out at 45°C for 25 minutes.

5. The method according to claim 1, wherein the steps are as follows: (1) Preparation of Fmoc-Gly-resin: Add 80 mmol of Fmoc-rink Amide AM resin to the synthesis reactor, wash with DCM, then wash with DMF, add 550 ml of prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, wash with DMF, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Gly-OH and 240 mmol of HOBT, add N,N-dimethylformamide and stir to dissolve, after complete dissolution, add 290 ml of 1 mol / L DIC / DMF solution, stir evenly and add to the reactor, stir and react at 30℃ for 24 hours, after the reaction is complete, remove the reaction solution, wash with DMF 3 times, then wash with DCM, and finally wash with methanol, dry, pour out, and put into a vacuum drying oven to dry for 12 h to obtain Fmoc-Gly-resin; (2) Preparation of Fmoc-Orn(Boc)-Gly-resin: Add the Fmoc-Gly-resin obtained in the previous step to the reaction vessel, wash with DCM, and then wash with DMF; add 550 ml of the prepared 30% piperidine / DMF solution to the reaction vessel, stir and react for 30 min, remove the reaction solution, wash with DMF, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Orn(Boc)-OH and 240 mmol of HOBT, add 400~500 ml of DMF and stir to dissolve, after complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reaction vessel, stir and react at 30℃ for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin; (3) Preparation of Fmoc-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Pro-OH and 240 mmol of HOBT, add 400~500 ml of DMF and stir to dissolve, after complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react at 30℃ for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin; (4) Preparation of Fmoc-Cys(Trt)-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Cys(Trt)-OH and 240 mmol of HOBT, add 400~500 ml of DMF and stir to dissolve, after complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react at 30℃ for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin; (5) Preparation of Fmoc-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Asn(Trt)-OH and 240 mmol of HOBT, add 400~500 ml of DMF and stir to dissolve, after complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react at 30℃ for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin; (6) Preparation of Fmoc-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, wash with DMF, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Thr(tBu)-OH and 240 mmol of HOBT, add 400~500 ml of DMF and stir to dissolve, after complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react at 30℃ for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin; (7) Preparation of Fmoc-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-Ile-OH and 240 mmol of HOBT, add 400~500 ml of DMF and stir to dissolve, after complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react at 30℃ for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin; (8) Preparation of Fmoc-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash, and the ninhydrin test should be positive; weigh 240 mmol of Fmoc-D-Tyr(Et)-OH and 240 mmol of HOBT, add 400~500 ml of DMF and stir to dissolve, after complete dissolution, add 290 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react at 30℃ for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin; (9) Preparation of Mpr(Trt)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-resin: Add 550 ml of the prepared 30% piperidine / DMF solution to the reactor, stir and react for 30 min, remove the reaction solution, add DMF to wash, and the ninhydrin test should be positive; weigh 320 mmol of Mpr(Trt)-OH and 320 mmol of HOBT, add 400~500 ml of DMF and stir to dissolve, after complete dissolution, add 385 ml of 1 mol / L DIC DMF solution and stir evenly; add the prepared amino acid coupling solution to the reactor, stir and react at 30℃ for 90 min, remove the reaction solution, wash with DMF, and dry to obtain the title resin, i.e., linear atosiban resin; (10) Cyclization: Weigh 162.6g of iodine and add it to a mixture of 5000ml of acetone and 25ml of formic acid. Stir and dissolve the mixture. After it is completely dissolved, add it to the reaction vessel and stir with nitrogen for 30min. After the reaction is complete, remove the reaction solution, wash with DMF, then wash with DCM, and finally wash with methanol. Dry the solution, pour it out, and vacuum dry it to obtain cyclized atosiban resin. (11) Pyrolysis: Add the cyclized resin obtained in the previous step to the reaction flask, add 2200 ml of the prepared pyrolysis reagent, stir and react for 120 min; filter, wash the resin with a small amount of trifluoroacetic acid, filter again; combine the filtrates, slowly settle into anhydrous ether; let stand at 5±2℃ for 2 h and then start centrifugation, wash with anhydrous ether after centrifugation, centrifuge again, and vacuum dry to obtain atosiban; the pyrolysis reagent is a mixture of trifluoroacetic acid: thioanisole: 1,2-ethylenedithiol: water in a ratio of 90%: 5%: 3%: 2%; (12) Purification: Dissolve the lysed atosiban in water, filter with a microporous membrane, load the filtrate onto a preparative high performance liquid chromatograph according to the purification conditions, collect the target peak solution, concentrate the collected solution, desalt, freeze dry to obtain purified atosiban; the purification conditions are: octadecylsilane bonded silica column, 10 μm, φ10cm×25cm; detection wavelength 230nm; flow rate 450ml / min; mobile phase A is 0.3% acetic acid aqueous solution, mobile phase B is acetonitrile; elution program: 0~10min maintain mobile phase B at 5%, 10~11min increase mobile phase B to 35%, 11~70min increase mobile phase B linearly to 45%, 70~71min increase mobile phase B to 50%, 71~75min maintain mobile phase B at 50%.

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