WO2018166146A1 - 一种多肽液相合成缩宫素的方法 - Google Patents

一种多肽液相合成缩宫素的方法 Download PDF

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WO2018166146A1
WO2018166146A1 PCT/CN2017/099882 CN2017099882W WO2018166146A1 WO 2018166146 A1 WO2018166146 A1 WO 2018166146A1 CN 2017099882 W CN2017099882 W CN 2017099882W WO 2018166146 A1 WO2018166146 A1 WO 2018166146A1
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compound
trt
acm
cys
oxytocin
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孙鹏程
胡碧煌
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LANZHOU KAIBO PHARMACEUTICAL Co Ltd
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LANZHOU KAIBO PHARMACEUTICAL Co Ltd
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Priority to EP17900782.8A priority patent/EP3438121B1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/16—Oxytocins; Vasopressins; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06043—Leu-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the preparation of a polypeptide medicine, in particular to a method for synthesizing oxytocin in a liquid phase, and belongs to the technical field of liquid phase synthesis of polypeptides.
  • Oxytocin also known as oxytocin, English name oxytocin, molecular formula C 43 H 66 N 12 O 12 S 2 , molecular weight of 1007.2, the chemical structural formula is as follows:
  • Oxytocin is mainly distributed in the posterior pituitary of the hypothalamus in humans and mammals. It is clinically used for oxytocin, postpartum hemorrhage and induction of labor. With the gradual deepening of its research, in addition to the common contraction of uterus, lactation and other effects of oxytocin, it has been found that oxytocin has many complex physiological functions, such as participation in learning, memory process, affecting drug addiction and Social adaptive behavior, maternal behavior, sexual behavior, feeding, pain regulation, and regulation of cardiovascular and body temperature.
  • the domestic oxytocin bulk drug is basically extracted from the posterior pituitary of pig or cattle hypothalamic pit with a titer of 160 IU/ml.
  • animal-derived products have biosafety risks, and at the same time, naturally occurring related impurities, such as It is difficult to be removed by purification such as vasopressin.
  • the liquid phase synthesis method of oxytocin involves safe and difficult to control reaction conditions (such as sodium ammonia method), many reaction steps, low yield; on the other hand, although the introduction of solid phase synthesis method simplifies oxytocin Synthetic process, however, from the literature reported at home and abroad, the yield, purity and potency of the synthesis of oxytocin by solid phase synthesis still cannot meet the demand.
  • the Boc solid phase synthesis method involves the dangerous compounds such as hydrogen fluoride in the industry. Not used, and Fmoc solid phase synthesis uses piperidine as a capping reagent. Piperidine is a liquid that is difficult to transport and store, especially as a regulated precursor chemical, which is inconvenient for the purchase, use and material management of the company.
  • the biggest challenge in the synthesis of oxytocin also includes the formation of disulfide bonds, the yield and purity of which cannot meet the needs of industrial production.
  • Other methods for deacetylation of a cysteine protecting group and cyclization to form a disulfide bond include, 1) after Photaki synthesizes a protected linear sequence of oxytocin, and then removes the thiol protecting group Bz or Z with sodium methoxide/methanol, and then The disulfide bond is formed by reaction with 1,2-diiodoethane, and finally the other protecting groups are removed by HBr/AcOH to obtain crude oxytocin.
  • the purified oxytocin has a titer of 380 IU/mg; Mühlemann also adopts 1, 2-diiodoethane acts as an oxidizing agent to form a disulfide bond, The yield of cyclization is 30%; 2) Fujii et al. used MBzl and Acm as the thiol protecting group to synthesize the protected oxytocin linear sequence, and stripped MBzl and Acm with trifluoroacetic acid strontium salt respectively to form two Sulfur bond, the crude product of oxytocin was obtained, the yield was 45% and 39% respectively; 3) Akaji et al.
  • oxytocin synthesis methods mainly include liquid phase method and Fmoc solid phase method.
  • the Chinese invention patent ZL2010102549195 discloses a method for synthesizing oxytocin in liquid phase: the method uses a sodium metal/liquid ammonia method to remove the protecting group, and then forms a disulfide bond by air oxidation, the yield is 25%, See biopotency reports.
  • Chinese invention patent ZL2008100849408 discloses a method for synthesizing oxytocin by liquid phase method and solid phase method: the method synthesizes two fragments protected by a Fmoc group of cysteine sulfhydryl groups, and performs fragment condensation (condensation time is 10) -40 hours), then, after the thiol protecting group Fmoc was removed with piperidine, a disulfide bond was formed by air oxidation.
  • the yield of the method is 7-10%, and the potency of the product is 600 IU/ml, and the purity thereof has not been reported.
  • the Chinese invention patent discloses a method for solid phase synthesis of oxytocin: the method uses Rink Amide resin as a solid phase carrier, Fmoc protects the amino acid as a monomer, and the cysteine adopts Fmoc-Cys (Trt).
  • Chinese invention patent discloses a method for solid phase synthesis of oxytocin: The method uses Rink Amide resin as the solid phase carrier, Fmoc protects the amino acid as monomer, and the N-terminal cysteine adopts Boc-Cys(Trt)-OH, and piperidine is used as the capping reagent, and the oxytocin is synthesized one by one.
  • oxytocin solid phase synthesis methods are: 1) the cyclization reaction is carried out in solution, and the disulfide bond is formed by air oxidation method or hydrogen peroxide oxidation method, the yield is up to 33%; 2) N-terminal The amino group is subjected to a cyclization reaction in an unprotected (free) state; 3) a piperidine is used for the capping reagent; 4) no bioavailability of oxytocin, or a reference bioavailability is reported.
  • the Boc polypeptide synthesis method and the Fmoc polypeptide synthesis method are used in combination, and all the reactions are carried out under mild conditions, and the sodium-sodium capping reaction reported in the literature of all liquid phase synthesis oxytocin at home and abroad is not used;
  • the use of highly toxic reagents or unsafe reaction conditions for the liquid phase synthesis of oxytocin greatly reduces the synthesis cost of oxytocin, and provides a reference for the industrial production of oxytocin.
  • All the chemical reactions in the present invention are carried out under mild conditions, and do not involve severely difficult to control reaction conditions and highly toxic chemical reagents, and obtain high purity (99% or more) and high titer (588 IU/mg,). Gongsu products.
  • the invention aims at the problems existing in the current oxytocin synthesis technology, and provides a liquid phase synthesis process of oxytocin which has high synthesis efficiency, low cost, environmental friendliness, mild reaction conditions, high purity and high titer, and can be industrially produced.
  • the finally obtained pure oxytocin was determined according to the European Pharmacopoeia (version 8.0), and the content of oxytocin produced by the present invention was 98.1%, and the titer was 588 IU/mg.
  • Fragment 1, fragment 2 and fragment 3 were synthesized separately, and then fragment 1 and fragment 2 were assembled to synthesize compound 4, and compound 4 was saponified.
  • Compound 5 was obtained, and compound 5 and fragment 3 were assembled to obtain the amino acid sequence of oxytocin (compound 6).
  • ethyl acetate was crystallized to obtain crude oxytocin, which was finally purified by C18 reverse phase silica gel chromatography. Oxytocin products.
  • reaction was carried out at 15-25 ° C, and after the reaction was completed by HPLC, the insoluble material was removed by filtration, concentrated, and solid was precipitated with 0.1 M hydrochloric acid, filtered, washed with water to neutral, and dried in vacuo to give compound 2.1.
  • the dried compound 2.1 was dissolved in an appropriate amount of diethylamine, concentrated at least in an amount, precipitated with petroleum ether, filtered, washed with petroleum ether and dried in vacuo to give compound 2.2.
  • the dried compound 2.5 was dissolved in an appropriate amount of diethylamine, concentrated to a minimum amount, and saturated NaHCO 3 was added, filtered, washed with water until neutral, dried under vacuum, then washed three times with petroleum ether, filtered, and dried in vacuo to give compound 2.6.
  • Fragment 1 (1.1 eq.), HOBt (1.1 eq.) was dissolved in THF and a small amount of DMF, cooled at -10 ° C for 10 min, DCC (1.1 eq.) was dissolved in a small amount of THF, added to the above solution, after 20 min reaction, weighed Fragment 2 (1 eq.) was dissolved in THF and added to the above reaction solution. The reaction was carried out at 15-25 ° C. After the reaction was completed by HPLC, the insoluble material was removed by filtration, concentrated, and the solid was precipitated with saturated sodium hydrogen carbonate solution, filtered, washed with water to neutral, and dried in vacuo to give compound 4.
  • Pretreatment of crude peptide 0.5 g of the crude peptide was weighed and dissolved in 10 ml of 0.1 M NaH 2 PO 4 solution, acetonitrile was added to make the concentration of acetonitrile 15%, and then filtered through a 0.45 ⁇ m microporous membrane.
  • the collected liquid with a purity of more than 99% is collected, concentrated at 25 ° C, desalted, concentrated, and lyophilized to obtain pure oxytocin.
  • the HPLC chromatogram is shown in the drawing; the European Pharmacopoeia standard is used as the standard substance, and according to the European Pharmacopoeia oxytocin content determination method, the oxytocin content obtained by the present invention is 98.1%, and the titer is 588 IU/mg.
  • the Boc polypeptide synthesis method and the Fmoc polypeptide synthesis method are used in combination, and all the reactions are carried out under mild conditions, and the sodium-sodium capping reaction reported in the literature of all liquid phase synthesis oxytocin at home and abroad is not used;
  • the use of highly toxic reagents or unsafe reaction conditions for the liquid phase synthesis of oxytocin greatly reduces the synthesis cost of oxytocin, and provides a reference for the industrial production of oxytocin.
  • All the chemical reactions in the present invention are carried out under mild conditions, and do not involve severely difficult to control reaction conditions and highly toxic chemical reagents, and obtain high purity (99% or more) and high titer (588 IU/mg). Prime product.
  • Figure 1 is a RP-HPLC chart of Fragment 1: Boc-Cys(Acm)-Tyr(tBu)-OH, analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 30-50% B, 0-30min, column: C18, 250x4.6mm, flow rate 1ml / min, detection wavelength 210nm;
  • Figure 2 is an ESI-MS diagram of the fragment 1 high acid cleavage sample H-Cys(Acm)-Tyr-OH;
  • Figure 3 is an RP-HPLC diagram of Fmoc-Cys(Acm)-Pro-OMe, analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 30-90% B, 0-30 min, column : C18, 250 x 4.6 mm, flow rate 1 ml / min, detection wavelength 210 nm;
  • Figure 4 is an RP-HPLC chart of H-Cys(Acm)-Pro-OMe, analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 0-10% B, 0-30 min, column : C18, 250 x 4.6 mm, flow rate 1 ml / min, detection wavelength 210 nm;
  • Figure 5 is an RP-HPLC chart of Fmoc-Asn(Trt)-Cys(Acm)-Pro-OMe, analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 50-90% B, 0 -30 min, column: C18, 250 x 4.6 mm, flow rate 1 ml / min, detection wavelength 210 nm;
  • Figure 6 is an RP-HPLC chart of H-Asn(Trt)-Cys(Acm)-Pro-OMe, analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 30-90% B, 0 -30 min, column: C18, 250 x 4.6 mm, flow rate 1 ml / min, detection wavelength 210 nm;
  • Figure 7 is an RP-HPLC chart of H-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe, analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 50- 90% B, 0-30 min, column: C18, 250 x 4.6 mm, flow rate 1 ml / min, detection wavelength 210 nm;
  • Figure 8 is an RP-HPLC chart of H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe (fragment 2), analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 50-90% B, 0-30 min, column: C18, 250 x 4.6 mm, flow rate 1 ml/min, detection wavelength 210 nm;
  • Figure 9 is an ESI-MS diagram of H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe (fragment 2);
  • Figure 10 is an RP-HPLC chart of Fmoc-Leu-Gly-NH2, analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 60-65% B, 0-30 min, column: C18, 250x4.6mm, flow rate 1ml/min, detection wavelength 210nm;
  • Figure 11 is an RP-HPLC chart of H-Leu-Gly-NH2 (fragment 3), analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 0-30% B, 0-30 min, chromatography Column: C18, 250 x 4.6 mm, flow rate 1 ml / min, detection wavelength 210 nm;
  • Figure 12 is an ESI-MS diagram of H-Leu-Gly-NH2 (fragment 3);
  • Figure 13 is a RP-HPLC chart of Compound 4, analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 75-80% B, 0-30 min, column: C18, 250 x 4.6 mm, flow rate 1ml/min, detection wavelength 210nm;
  • Figure 14 is a RP-HPLC chart of Compound 5, analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 80-90% B, 0-30 min, column: C18, 250 x 4.6 mm, flow rate 1ml/min, detection wavelength 210nm;
  • Figure 15 is a RP-HPLC chart of Compound 6, analysis conditions: A: 3% ACN / H2O, 0.1% TFA, B: ACN, 80-85% B, 0-30 min, column: C18, 250 x 4.6 mm, flow rate 1ml/min, detection wavelength 210nm;
  • Figure 16 is a RP-HPLC chart of Compound 7, analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 80-85% B, 0-30 min, column: C18, 250 x 4.6 mm, flow rate 1ml/min, detection wavelength 210nm;
  • Figure 17 is an RP-HPLC diagram of crude oxytocin, analysis conditions: A: 0.1 M NaH2PO4/H2O, B: 50% CAN/H2O, 30-60% B, 0-30 min, column: C18, 250 x 4.6 mm , flow rate 1ml / min, detection wavelength 220nm;
  • Figure 18 is an RP-HPLC chart of pure oxytocin, analysis conditions: A: 0.1M NaH2PO4/H2O, B: 50% CAN/H2O, 30-60% B, 0-30 min, column: C18, 250x4. 6mm, flow rate 1ml/min, detection wavelength 220nm;
  • Figure 19 is an ESI-MS diagram of pure oxytocin
  • Figure 20 is an HPLC chart of the product Boc-Leu-Gly-NH2 synthesized according to Scheme 1 of the fragment 3, analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 10-50% B, 0-30 min , column: C18, 250x4.6mm, flow rate 1ml / min, detection wavelength 210nm;
  • Figure 21 is an HPLC chart of the product H-Leu-Gly-NH2 synthesized according to Scheme 1 of the fragment 3, analysis conditions: A: 3% ACN/H2O, 0.1% TFA, B: ACN, 0-30% B, 0-30 min Column: C18, 250 x 4.6 mm, flow rate 1 ml/min, detection wavelength 210 nm.
  • Boc-Cys(Acm)-OH (100 mol, 29.2 g, 1 eq.), HOSu (110 mmol, 12.6 g, 1.1 eq.) was dissolved in 200 ml of tetrahydrofuran, and then cooled to 10 ° C for 10 min, DCC (110 mmol, 22.7 g, 1.1 eq.) was dissolved in 20 ml of THF and added to the above solution. After 20 min of reaction, H-Tyr(tBu)-OH (110 mmol, 26.1 g, 1.1 eq.) and NaHCO 3 (110 mmol, 9.2 g, 1.1 eq.) were weighed.
  • Fmoc-Cys(Acm)-OH (100 mmol, 41.5 g, 1 eq.), HOBt (105 mmol, 14.1 g, 1.05 eq.), was dissolved in 200 ml of THF and 30 ml of DMF, and cooled to 10 ° C for 10 min, DCC (105 mmol, 21.6 g, 1.05 eq.) was dissolved in 20 ml of THF and added to the above solution. After reacting for 20 min, H-Pro-OMe.HCl (105 mmol, 17.4 g, 1.05 eq.) was dissolved in 100 ml of THF and added to TEA (105 mmol, 14.5).
  • the dried compound 2.1 was dissolved in 200 ml of diethylamine, concentrated at least in an amount, precipitated with petroleum ether, filtered, washed with petroleum ether and dried in vacuo to give compound 2.2.
  • the purity was 98.0%, the product was 28.8 g, and the yield was 95%.
  • the HPLC analysis pattern is shown in Figure 4 of the specification.
  • the dried compound 2.3 was dissolved in 200 ml of diethylamine, concentrated to a minimum amount, and saturated NaHCO 3 was added, filtered, washed with water to neutral, dried in vacuo, then washed three times with petroleum ether, filtered, and dried in vacuo to give compound 2.4.
  • the purity was 96.3%, the product was 55.7 g, and the yield was 84.4%.
  • the HPLC analysis pattern is shown in Figure 6 of the specification.
  • the dried compound 2.5 was dissolved in 150 ml of diethylamine, concentrated at least, added with saturated NaHCO 3 , filtered, washed with water until neutral, dried under vacuum, then washed three times with petroleum ether, filtered, dried in vacuo to give compound 2.6, purity 98.3%, product 86.5 g, yield 84%.
  • the HPLC analysis pattern is shown in Figure 7 of the specification.
  • the dried compound 2.7 was dissolved in 150 ml of diethylamine, concentrated to a minimum amount, and saturated NaHCO 3 was added, filtered, washed with water until neutral, dried under vacuum, then washed three times with petroleum ether, filtered and dried in vacuo to give compound 2.
  • the purity was 94.2%, the product was 91.5 g, and the yield was 80.1%.
  • the HPLC analysis pattern is shown in Figure 8 of the specification.
  • the ESI-MS spectrum of the high acid cleavage sample H-Ile-Gln-Asn-Cys(Acm)-Pro-OMe m/z calculated.658.3; found 681.1.3[M+Na] + ) is shown in Figure 9. .
  • Fragment 1 (1.1 eq., 88 mmol, 45 g), HOBt (1.1 eq., 88 mmol, 11.9 mg) was dissolved in 150 ml of THF and 60 ml of DMF, and cooled to 10 ° C for 10 min, DCC (1.1 eq., 88 mmol, 18.1 mg) 20 ml of THF was dissolved and added to the above solution. After reacting for 20 min, the fragment 2 (1 eq., 80 mmol, 91.5 g) was dissolved in 100 ml of THF and added to the above reaction solution. The reaction was carried out at 15-25 ° C.
  • Pretreatment of crude peptide 0.5 g of the crude peptide was weighed and dissolved in 10 ml of 0.1 M NaH 2 PO 4 solution, acetonitrile was added to make the concentration of acetonitrile 15%, and then filtered through a 0.45 ⁇ m microporous membrane.
  • the collected liquid with a purity of more than 99% was collected, concentrated at 25 ° C, the PS polymer was desalted, acetic acid was added, concentrated, and lyophilized to obtain 10.6 g of oxytocin.
  • the HPLC spectrum is shown in Fig. 18, and the ESI-MS chart is shown in Fig. 19 (oxytocin). m/z calculated.1006.4; found1007.5[M+H] + ); using the European Pharmacopoeia standard as the standard substance, according to the European Pharmacopoeia oxytocin content determination method, removing water and acetic acid, the content is 98.2%, the titer is 588 IU/mg.

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Abstract

一种反应条件温和的液相合成缩宫素的方法，该方法合成了三个缩宫素片段：片段1：Boc-Cys(Acm)-Tyr(tBu)-OH，片段2：H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe，片段3：H-Leu-Gly-NH 2，将片段组装合成全保护的缩宫素氨基酸序列后，用碘脱去Acm并同时环化形成二硫键得到保护的环状缩宫素，最后用三氟乙酸除去剩余的保护基得到缩宫素粗品，用乙酸乙酯重结晶后，经反相色谱纯化得到高纯度(粗品纯度达95％)和高效价的缩宫素产品(588 IU/mg)。该方法结合使用了Boc多肽合成法和Fmoc多肽合成法，所有反应均在温和条件下进行，未使用氨钠法脱帽反应；未使用剧毒试剂或不安全的反应条件进行缩宫素的液相合成，极大降低了缩宫素的合成成本，为工业化生产缩宫素提供了方法。

Description

一种多肽液相合成缩宫素的方法 技术领域
本发明涉及多肽类药物的制备，具体其涉及一种液相合成缩宫素的方法，属于多肽液相合成技术领域。
背景技术
缩宫素，也叫催产素，英文名oxytocin，分子式C43H66N12O12S2，分子量为1007.2，化学结构式如下：
Figure PCTCN2017099882-appb-000001
缩宫素主要分布在人和哺乳动物的下丘脑垂体后叶，临床上用于催产、产后出血和引产。随着对其研究的逐渐深入，除了缩宫素常见的收缩子宫、泌乳等作用外，人们发现缩宫素还有许多复杂的生理功能，如参与学习、记忆过程、影响药物成瘾性及对社会适应性行为、母性行为、性行为、摄食、痛觉调节以及心血管、体温的调节等。
目前，国内的缩宫素原料药基本上自猪或牛下丘脑垂体后叶中提取，效价160IU/ml，而且，动物来源的产品存在生物安全性风险，同时，天然存在的相关杂质，如加压素等，难以被纯化除去。
迄今为止，缩宫素的液相合成法涉及安全难以掌控的反应条件(如氨钠法)，反应步骤多，产率低；另一方面，虽然固相合成法的引入简化了缩宫素的合成工艺，但是，从国内外报道的文献来看，固相合成法合成缩宫素的产率、纯度和效价仍不能满足需求，其中Boc固相合成法涉及氟化氢等危险化合物在工业上逐渐不被采用，而Fmoc固相合成法均采用哌啶作为脱帽试剂。哌啶是一种液体，不易运输和储存，特别是作为受管制的易制毒化学品，对企业的采购、使用和物料管理带来不便。
缩宫素合成的最大的挑战还包括，二硫键的形成，其产率和纯度，不能满足工业生产的需求。
1953年，Vincent du Vigneaud首次用多肽液相合成法合成了缩宫素，并证实了其生物活性；随后又将此方法进行了改进。这些两种方法最终均采用金属钠/液氨脱去保护缩宫素的保护基和空气氧化法形成二硫键。其他的脱半胱氨酸保护基和环化形成二硫键的方法，包括，1)Photaki合成了保护的缩宫素直链序列后，用甲醇钠/甲醇脱去巯基保护基Bz或Z，然后用1,2-二碘乙烷反应形成二硫键，最后用HBr/AcOH脱去其他保护基得到缩宫素粗品，其纯化所得的缩宫素效价380IU/mg；Mühlemann也采用了1,2-二碘乙烷作为氧化剂形成二硫键， 环化收率30％；2)Fujii等分别选用MBzl和Acm为巯基保护基的半胱氨酸合成了保护的缩宫素直链序列，用三氟乙酸铊盐分别脱去MBzl和Acm并形成二硫键，得到缩宫素粗品，产率分别为45％和39％；3)Akaji等分别选用Acm,Tacm和But为巯基保护基的半胱氨酸合成了保护的缩宫素直链序列，用甲基三氯硅烷/二苯基亚砜分别脱去了Acm,Tacm,和But并形成二硫键，环化产率分别是56％，69％和64％。Fernando Albericio等用Boc/MBHA resin法和Fmoc/PAL resin法进行了缩宫素固相合成的比较研究，还对形成二硫键的方法进行了研究，包括空气氧化法、碘氧化法、三氟乙酸铊盐氧化法。二硫键形成的研究结果表明，三氟乙酸铊盐的效果最佳(产率72％)，碘氧化法的产率28％。
目前，国内报道的缩宫素合成方法主要有液相法和Fmoc固相法。其中，中国发明专利ZL2010102549195公布了一种液相合成缩宫素的方法:该方法用金属钠/液氨法脱去保护基，然后，用空气氧化法形成二硫键，收率25％，未见生物效价报道。中国发明专利ZL2008100849408公布了一种液相法和固相法结合合成缩宫素的方法:该方法合成了半胱氨酸巯基用Fmoc基团保护的两个片段，进行片段缩合(缩合时间为10-40小时)，然后，用哌啶将巯基保护基Fmoc脱去后，采用空气氧化法形成二硫键。该方法收率7-10％，产品的效价为600IU/ml，未见报道其纯度。这里用体积计算效价的方法，无法判断缩宫素的真实效价，因为每毫升所含缩宫素的量未知。中国发明专利(专利号ZL2005101123565)公布了一种固相合成缩宫素的方法:该方法以Rink Amide树脂为固相载体，Fmoc保护氨基酸为单体，其中半胱氨酸采用Fmoc-Cys(Trt)-OH，使用哌啶作为脱帽试剂，依次逐个合成了保护缩宫素氨基酸序列，然后，将肽链从树脂上切割下来，乙醚沉淀切肽溶液获得还原性缩宫素后，在偏碱性溶液中(pH7-8)用空气、或双氧水进行氧化，形成二硫键，收率21％，未见报道效价；中国发明专利(ZL201210256922X)公布了一种固相合成缩宫素的方法:该方法以Rink Amide树脂为固相载体，Fmoc保护氨基酸为单体，其中N末端半胱氨酸采用Boc-Cys(Trt)-OH，使用哌啶作为脱帽试剂，依次逐个合成了保护缩宫素氨基酸序列，然后，将肽链从树脂上切割下来，乙醚沉淀切肽溶液获得未环化的缩宫素后，在偏碱性溶液中用空气、或双氧水进行氧化，形成二硫键，其收率33％，未见报道效价。这些缩宫素固相合成法的共同点是：1)环化反应均在溶液中进行，均采用空气氧化法、或双氧水氧化法形成二硫键，收率最高为33％；2)N末端氨基在未保护(游离)状态下进行环化反应；3)脱帽试剂均采用哌啶；4)未见报道缩宫素的生物效价、或可参考的生物效价。
综上所述，目前国内外缩宫素的合成还存在一系列的挑战，如使用易制毒化学品哌啶、乙醚等，剧毒且昂贵的试剂三氟乙酸铊盐等，难以掌控的反应条件(如金属钠/液氨、HF等)，而且，合成所得缩宫素产品的收率和效价都较低。
在本发明中，结合使用了Boc多肽合成法和Fmoc多肽合成法，所有反应均在温和条件下进行，未使用国内外所有液相合成缩宫素的文献报道的氨钠法脱帽反应；首次未使用剧毒试剂或不安全的反应条件进行缩宫素的液相合成，极大降低了缩宫素的合成成本，为工业化生产缩宫素提供了参考。本发明中所有的化学反应均在温和的条件下进行，不涉及剧烈难以控制的反应条件和剧毒的化学试剂，得到了高纯度(99％以上)、高效价(588IU/mg，)的缩宫素产品。
发明内容
本发明针对目前缩宫素合成技术存在的问题，提供一种合成效率高、成本较低、环境友好、反应条件温和、纯度和效价高，可以工业化生产的缩宫素液相合成工艺。最终所得的缩宫素纯品，按照欧洲药典(8.0版)，含量测定法测定本发明所生产的缩宫素含量为98.1％，效价588IU/mg。
本发明的方法步骤如下：
分别合成片段1、片段2和片段3，然后片段1和片段2组装合成化合物4，化合物4皂化后 得到化合物5，化合物5和片段3组装得到保护缩宫素氨基酸序列(化合物6)，经环化和脱帽反应后，乙酸乙酯结晶得到缩宫素粗品，最后采用C18反相硅胶色谱纯化，得到缩宫素产品。
按照本发明优选的方案：
1.合成片段1：Boc-Cys(Acm)-Tyr(tBu)-OH
将Boc-Cys(Acm)-OH(1eq.),HOSu(1.1eq.),用四氢呋喃溶解，-10℃冷浴10min，DCC(1.1eq.)用少量THF溶解，加入以上溶液中，反应20min后，称H-Tyr(tBu)-OH(1.1eq.)和NaHCO3(1.1eq.)用水溶解，加入以上溶液中。15-25℃反应，HPLC监测反应完成后，0.5M盐酸调pH至中性，过滤掉不溶物，浓缩除去THF，0.5M盐酸调pH至2得白色固体，过滤，水洗至中性，真空干燥得片段1。
2.合成片段2：H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe
(1)合成化合物2.1：Fmoc-Cys(Acm)-Pro-OMe：
将Fmoc-Cys(Acm)-OH(1eq.),HOBt(1.1eq.),用THF和少量DMF溶解，-10℃冷浴10min，DCC(1.1eq.)用少量THF溶解，加入以上溶液中，反应20min后，称H-Pro-OMe.HCl(1.1eq.)用THF溶解，加入TEA(1.1eq.)，混匀，加入以上反应溶液中。15-25℃反应，HPLC监测反应完全后，过滤除去不溶物，浓缩，用0.1M盐酸析出固体，过滤，水洗至中性，真空干燥得化合物2.1。
(2)合成化合物2.2：H-Cys(Acm)-Pro-OMe：
将干燥好的化合物2.1用适量二乙胺溶解，浓缩至少量，加石油醚析出，过滤，石油醚洗涤，真空干燥得化合物2.2。
(3)合成化合物2.3：Fmoc-Asn(Trt)-Cys(Acm)-Pro-OMe：
将Fmoc-Asn(Trt)-OH(1.1eq.),HOBt(1.1eq.),用THF和少量DMF溶解，-10℃冷浴10min，DCC(1.1eq.)用少量THF溶解，加入以上溶液中，反应20min后，称化合物2.2(1eq.)用THF溶解，加入以上反应溶液中。15-25℃反应，HPLC监测反应完全后，过滤除去不溶物，浓缩，用0.1M盐酸析出固体，过滤，水洗至中性，真空干燥得化合物2.3。
(4)合成化合物2.4：H-Asn(Trt)-Cys(Acm)-Pro-OMe：
将干燥好的化合物2.3用适量二乙胺溶解，浓缩至少量，加饱和NaHCO3析出，过滤，水洗至中性，真空干燥，然后用石油醚搅拌洗涤三次，过滤，真空干燥得化合物2.4。
(5)合成化合物2.5：Fmoc-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe：
将Fmoc-Gln(Trt)-OH(1.1eq.),HOBt(1.1eq.),用THF和少量DMF溶解，-10℃冷浴10min，DCC(1.1eq.)用少量THF溶解，加入以上溶液中，反应20min后，称化合物2.4(1eq.)用THF溶解，加入以上反应溶液中。15-25℃反应，HPLC监测反应完全后，过滤除去不溶物，浓缩，用0.1M盐酸析出固体，过滤，水洗至中性，真空干燥得化合物2.5。
(6)合成化合物2.6：H-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe：
将干燥好的化合物2.5用适量二乙胺溶解，浓缩至少量，加饱和NaHCO3析出，过滤，水洗至中性，真空干燥，然后用石油醚搅拌洗涤三次，过滤，真空干燥得化合物2.6。
(7)合成化合物2.7：Fmoc-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe：
将Fmoc-Ile-OH(1.1eq.),HOBt(1.1eq.),用THF和少量DMF溶解，-10℃冷浴10min， DCC(1.1eq.)用少量THF溶解，加入以上溶液中，反应20min后，称化合物2.6(1eq.)用THF溶解，加入以上反应溶液中。15-25℃反应，HPLC监测反应完全后，过滤除去不溶物，浓缩，用0.1M盐酸析出固体，过滤，水洗至中性，真空干燥得化合物2.7。
(8)合成片段2：H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe：
将干燥好的化合物2.7用适量二乙胺溶解，浓缩至少量，加饱和NaHCO3析出，过滤，水洗至中性，真空干燥，然后用石油醚搅拌洗涤三次，过滤，真空干燥得化合物2。
3.合成片段3：H-Leu-Gly-NH2
称Fmoc-Leu-OH(1eq.),BOP(1eq.)用DMF溶解，加DIPEA(1.2eq.)，5min后，称H-Gly-NH2.HCl(1.1eq.)用DMF溶解，加TEA(1.1eq.)，倒入以上反应液中，TLC(DCM:MeOH:AcOH＝100:6:1)跟踪监测,反应完全后，用0.1M HCl溶液析出，水洗至中性，真空干燥得产物Fmoc-Ile-Gly-NH2.HCl。将干燥的Fmoc-Ile-Gly-NH2.HCl加入适量二乙胺，TLC(DCM:MeOH:AcOH＝100:6:1)跟踪监测，反应完全后，浓缩，石油醚析出，过滤，石油醚洗涤，真空干燥得到片段3。
4.合成化合物4：
称片段1(1.1eq.),HOBt(1.1eq.)用THF和少量DMF溶解，-10℃冷浴10min，DCC(1.1eq.)用少量THF溶解，加入以上溶液中，反应20min后，称片段2(1eq.)用THF溶解，加入以上反应溶液中。15-25℃反应，HPLC监测反应完全后，过滤除去不溶物，浓缩，用饱和碳酸氢钠溶液析出固体，过滤，水洗至中性，真空干燥得化合物4。
5.合成化合物5：
化合物4(1eq.)用THF溶解，-10℃冷浴10min，加入2M LiOH(5eq.)溶液冰浴反应，HPLC检测反应完全后，0.5M盐酸溶液调pH至中性，浓缩，析出白色固体，过滤，水洗，真空干燥得化合物5。
6.合成化合物6：
化合物5(1eq.)、BOP(1eq.)用DMF溶解，加入DIPEA(1.1eq.)，5min后，称片段3(1.1eq.)用DMF溶解，投入以上反应液。HPLC检测反应完全后，0.1M盐酸溶液析出白色固体，过滤，水洗至中性，真空干燥得化合物5。
7.合成化合物7：
碘(10eq.)用DMF溶解，缓慢加入化合物6(1eq.)，HPLC检测反应完全后，0.1％硫代硫酸钠溶液析出白色固体，过滤，水洗，真空干燥得化合物7。
8.合成缩宫素粗品：
将化合物7溶于三氟乙酸，10min后，浓缩至少量，加DCM，浓缩至少量，重复三次，加乙酸乙酯冷藏，过夜，过滤，固体真空干燥。
9.缩宫素粗品纯化：
粗肽的预处理：称取0.5g粗肽用10ml 0.1M NaH2PO4溶液溶解，加入乙腈使得乙腈的浓度为15％，然后用0.45μm微孔滤膜过滤。
纯化条件与梯度：
制备柱：DAC HB-50，富士硅胶
Figure PCTCN2017099882-appb-000002
-10μm-C18
检测波长：220nm，流速：50ml/min
流动相：A 0.1M NaH2PO4溶液，B 50％乙腈/A，0-60min：30％B-50％B
收集纯度99％以上的收集液，25℃浓缩，PS聚合物脱盐，浓缩，冻干，得缩宫素纯品， HPLC图谱见附图；以欧洲药典标准品为标准物质，按欧洲药典缩宫素含量测定法，本发明所得到的缩宫素含量为98.1％，效价588IU/mg。
本发明的有益效果：
在本发明中，结合使用了Boc多肽合成法和Fmoc多肽合成法，所有反应均在温和条件下进行，未使用国内外所有液相合成缩宫素的文献报道的氨钠法脱帽反应；首次未使用剧毒试剂或不安全的反应条件进行缩宫素的液相合成，极大降低了缩宫素的合成成本，为工业化生产缩宫素提供了参考。本发明中所有的化学反应均在温和的条件下进行，不涉及剧烈难以控制的反应条件和剧毒的化学试剂，得到了高纯度(99％以上)、高效价(588IU/mg)的缩宫素产品。
附图说明：
图1是片段1：Boc-Cys(Acm)-Tyr(tBu)-OH的RP-HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,30-50％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图2是片段1高酸裂解样品H-Cys(Acm)-Tyr-OH的ESI-MS图；
图3是Fmoc-Cys(Acm)-Pro-OMe的RP-HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,30-90％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图4是H-Cys(Acm)-Pro-OMe的RP-HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,0-10％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图5是Fmoc-Asn(Trt)-Cys(Acm)-Pro-OMe的RP-HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,50-90％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图6是H-Asn(Trt)-Cys(Acm)-Pro-OMe的RP-HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,30-90％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图7是H-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe的RP-HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,50-90％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图8是H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe(片段2)的RP-HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,50-90％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图9是H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe(片段2)的ESI-MS图；
图10是Fmoc-Leu-Gly-NH2的RP-HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,60-65％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图11是H-Leu-Gly-NH2(片段3)的RP-HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,0-30％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图12是H-Leu-Gly-NH2(片段3)的ESI-MS图；
图13是化合物4的RP-HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,75-80％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图14是化合物5的RP-HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,80-90％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图15是化合物6的RP-HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,80-85％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图16是化合物7的RP-HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,80-85％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图17是缩宫素粗品的RP-HPLC图，分析条件：A:0.1M NaH2PO4/H2O,B:50％CAN/H2O,30-60％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长220nm；
图18是缩宫素纯品的RP-HPLC图，分析条件：A:0.1M NaH2PO4/H2O,B:50％CAN/H2O,30-60％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长220nm；
图19是缩宫素纯品的ESI-MS图；
图20是片段3按方案1合成的产物Boc-Leu-Gly-NH2的HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,10-50％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm；
图21是片段3按方案1合成的产物H-Leu-Gly-NH2的HPLC图，分析条件：A:3％ACN/H2O,0.1％TFA,B:ACN,0-30％B,0-30min,色谱柱：C18,250x4.6mm,流速1ml/min,检测波长210nm。
具体实施方式
在本发明权利要求书和说明书中出现物质的英文缩写对应的中文名称见表1
英文缩写 中文名称
Fmoc 9-芴甲氧羰基
Trt 三苯甲基
HOBt 1-羟基苯并三唑
Boc 叔丁氧羰基
tBu 叔丁基
TFA 三氟乙酸
TIS 三异丙基硅烷
DIPEA N,N-二异丙基乙胺
IPA 异丙醇
DMF N,N-二甲基甲酰胺
Gly 甘氨酸
Leu 亮氨酸
Pro 脯氨酸
Asn 门冬酰胺
Gln 谷氨酰胺
Cys 半胱氨酸
Ile 异亮氨酸
Tyr 酪氨酸
THF 四氢呋喃
DCC 二环己基碳二亚胺
HOOBt 3-羟基-1,2,3-苯并三嗪-4(3H)-酮
TEA 三乙胺
DEA 二乙胺
HOSu N-羟基琥珀酰亚胺
表1 本发明中出现物质的英文缩写对应的中文名称
实施例1：
合成片段1：Boc-Cys(Acm)-Tyr(tBu)-OH
将Boc-Cys(Acm)-OH(100mol,29.2g,1eq.),HOSu(110mmol,12.6g,1.1eq.)，用四氢呋喃200ml溶解，-10℃冷浴10min，DCC(110mmol,22.7g,1.1eq.)用20ml THF溶解，加入以上溶 液中，反应20min后，称H-Tyr(tBu)-OH(110mmol,26.1g,1.1eq.)和NaHCO3(110mmol,9.2g,1.1eq.)用150ml水溶解，加入以上溶液中。15-25℃反应，HPLC监测反应完成后，0.5M盐酸调pH至中性，过滤掉不溶物，浓缩除去THF，0.5M盐酸调pH至2得白色固体，过滤，水洗至中性，真空干燥得片段1，纯度99.1％，产物47.1g，收率92％。片段1：Boc-Cys(Acm)-Tyr(tBu)-OH RP-HPLC图谱见说明书附图1，其高酸裂解样品(H-Cys(Acm)-Tyr-OH m/z calculated.355.4；found 356.3[M+H]+)的ESI-MS质谱图见附图2。
实施例2：
合成片段2：H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe
(1)合成化合物2.1：Fmoc-Cys(Acm)-Pro-OMe：
将Fmoc-Cys(Acm)-OH(100mmol,41.5g,1eq.),HOBt(105mmol,14.1g,1.05eq.)，用200ml THF和30ml DMF溶解，-10℃冷浴10min，DCC(105mmol,21.6g,1.05eq.)用20ml THF溶解，加入以上溶液中，反应20min后，称H-Pro-OMe.HCl(105mmol,17.4g,1.05eq.)用100ml THF溶解，加入TEA(105mmol,14.5ml,1.05eq.)，混匀，加入以上反应溶液中。15-25℃反应，HPLC监测反应完全后，过滤除去不溶物，浓缩，用0.1M盐酸析出固体，过滤，水洗至中性，真空干燥得化合物2.1，纯度95.3％，产物50.9g，收率97％(产率均按100mmol计算，下同)。HPLC分析图谱见说明书附图3。
(2)合成化合物2.2：H-Cys(Acm)-Pro-OMe：
将干燥好的化合物2.1用200ml二乙胺溶解，浓缩至少量，加石油醚析出，过滤，石油醚洗涤，真空干燥得化合物2.2。纯度98.0％，产物28.8g，产率95％。HPLC分析图谱见说明书附图4。
(3)合成化合物2.3：Fmoc-Asn(Trt)-Cys(Acm)-Pro-OMe：
将Fmoc-Asn(Trt)-OH(1.1eq.,104.4mmol,62.3g),HOBt(1.1eq.,104.4mmol,14.1g),用DCM200ml和DMF20ml溶解，-10℃冷浴10min，DCC(1.1eq.,104.4mmol,21.5g)用20ml DCM溶解，加入以上溶液中，反应20min后，称化合物2.2(1eq.94.9mmol,28.8g)用50ml DMF溶解，加入以上反应溶液中。15-25℃反应，HPLC监测反应完全后，过滤除去不溶物，旋转蒸发仪浓缩，用0.1M盐酸析出固体，过滤，水洗至中性，真空干燥得化合物2.3。纯度99％，产物75g，产率85％。HPLC分析图谱见说明书附图5。
(4)合成化合物2.4：H-Asn(Trt)-Cys(Acm)-Pro-OMe：
将干燥好的化合物2.3用200ml二乙胺溶解，浓缩至少量，加饱和NaHCO3析出，过滤，水洗至中性，真空干燥，然后用石油醚搅拌洗涤三次，过滤，真空干燥得化合物2.4。纯度96.3％，产物55.7g，产率84.4％。HPLC分析图谱见说明书附图6。
(5)合成化合物2.5：Fmoc-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe：
将Fmoc-Gln(Trt)-OH(1.1eq.,92.8mmol,56.7g),HOBt(1.1eq.,92.8mmol,12.5g),用150ml THF和50ml DMF溶解，-10℃冷浴10min，DCC(1.1eq.,92.8mmol,19.1g)用20ml THF溶解，加入以上溶液中，反应20min后，称化合物2.4(1eq.,84.4mmol,55.7g)用150ml THF溶解，加入以上反应溶液中。15-25℃反应，HPLC监测反应完全后，过滤除去不溶物，浓缩，用0.1M盐酸析出固体，过滤，水洗至中性，真空干燥得化合物2.5,产物106.4g，产率85％。
(6)合成化合物2.6：H-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe：
将干燥好的化合物2.5用150ml二乙胺溶解，浓缩至少量，加饱和NaHCO3析出，过滤，水洗至中性，真空干燥，然后用石油醚搅拌洗涤三次，过滤，真空干燥得化合物2.6,纯度98.3％，产物86.5g，产率84％。HPLC分析图谱见说明书附图7。
(7)合成化合物2.7：Fmoc-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe：
将Fmoc-Ile-OH(1.1eq.,92.4mmol,32.6g),HOBt(1.1eq.,92.4mmol,12.5g),用150ml THF和50ml DMF溶解，-10℃冷浴10min，DCC(1.1eq.,92.4mmol,19g)用少20ml THF溶解，加入以上溶液中，反应20min后，称化合物2.6(1eq.,84mmol,86.5g)用150ml THF溶解，加入以上反应溶液中。15-25℃反应，HPLC监测反应完全后，过滤除去不溶物，浓缩，用0.1M盐酸析出固体，过滤，水洗至中性，真空干燥得化合物2.7。纯度90.3％，产物114.4g，产率83.8％。
(8)合成片段2：H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe：
将干燥好的化合物2.7用150ml二乙胺溶解，浓缩至少量，加饱和NaHCO3析出，过滤，水洗至中性，真空干燥，然后用石油醚搅拌洗涤三次，过滤，真空干燥得化合物2。纯度94.2％，产物91.5g，产率80.1％。HPLC分析图谱见说明书附图8。其高酸裂解样品(H-Ile-Gln-Asn-Cys(Acm)-Pro-OMe m/z calculated.658.3；found 681.1.3[M+Na]+)的ESI-MS质谱图见附图9。
实施例3：
按方案2合成片段3：H-Leu-Gly-NH2
称Fmoc-Leu-OH(1eq.,100mmol,35.3g),BOP(1.01eq.,101mmol,44.7g)用DMF 200ml溶解，加DIPEA(1.2eq.,120mmol,21ml)，5min后，称H-Gly-NH2.HCl(1.1eq.,110mmol,12.2g)用DMF100ml溶解，加TEA(1.1eq.,110mmol,15ml)，倒入以上反应液中，TLC(DCM:MeOH:AcOH＝100:6:1)跟踪监测,反应完全后，用0.1M HCl溶液析出，水洗至中性，真空干燥得产物Fmoc-Leu-Gly-NH2.HCl,纯度97％，产物43.7g，产率98％,HPLC分析图谱见说明书附图10。
将干燥的Fmoc-Leu-Gly-NH2.HCl加入400ml二乙胺，TLC(DCM:MeOH:AcOH＝100:6:1)跟踪监测，反应完全后，浓缩，石油醚析出，过滤，石油醚洗涤，真空干燥得到片段3,纯度98.4％，产物17.9g，产率95.6％,HPLC分析图谱见说明书附图11，(H-Leu-Gly-NH2m/z calculated.187.1；found 210.0[M+Na]+)ESI-MS质谱图见附图12。
实施例4：
合成化合物4：
称片段1(1.1eq.,88mmol,45g),HOBt(1.1eq.,88mmol,11.9mg)用THF150ml和DMF 60ml溶解，-10℃冷浴10min，DCC(1.1eq.,88mmol,18.1mg)用20ml THF溶解，加入以上溶液中，反应20min后，称片段2(1eq.,80mmol,91.5g)用THF100ml溶解，加入以上反应溶液中。15-25℃反应，HPLC监测反应完全后，过滤除去不溶物，浓缩，用饱和碳酸氢钠溶液析出固体，过滤，水洗至中性，真空干燥得化合物4。纯度86％，产物115.5g，产率70.6％,RP-HPLC分析图谱见说明书附图13。
实施例5：
合成化合物5：
化合物4(1eq.,115.5g)用THF 800ml溶解，-10℃冷浴10min，加入1.5M NaOH(5eq.,353mmol)溶液235ml冰浴反应，HPLC检测反应完全后，0.5M盐酸溶液调pH至中性，浓缩，析出白色固体，过滤，水洗，真空干燥得化合物5。纯度93％，产物106.2g，产率65.5％,RP-HPLC分析图谱见说明书附图14。
实施例6：
合成化合物6：
化合物5(1eq.,65.5mmol,106.2g)、BOP(1.05eq.,68.8mmol,30.4g)用DMF150ml溶解，加入DIPEA(1.1eq.,72mmol,12.5ml)，5min后，称片段3(1.1eq.,72mmol,13.4g)用DMF100ml溶解，投入以上反应液。HPLC检测反应完全后，0.1M盐酸溶液析出白色固体，过滤，水洗至中性，真空干燥得化合物5。纯度88.6％，产物107.5g，产率60％,RP-HPLC分析图谱见说明书附图15。
实施例7：
合成化合物7：
碘(10eq.,600mmol,152g)用DMF 1200ml溶解，缓慢加入化合物6(1eq.,60mmol,107.5g)，HPLC检测反应完全后，0.1％硫代硫酸钠溶液析出白色固体，过滤，水洗，真空干燥得化合物7。纯度95％，产物96.5g，产率58.6％,RP-HPLC分析图谱见说明书附图16。
实施例8：
合成缩宫素粗品：
将化合物7溶于三氟乙酸裂解液(TFA:TIS:H2O＝95:5:5)200ml，5min后，浓缩至少量，加DCM，浓缩至少量，重复三次，加乙酸乙酯冷藏，10h后，过滤，固体真空干燥。得到缩宫素粗品，纯度94.5％，产物称重26.6g，产率26.4％，RP-HPLC分析图谱见说明书附图17。
实施例9：
缩宫素粗品纯化：
粗肽的预处理：称取0.5g粗肽用10ml 0.1M NaH2PO4溶液溶解，加入乙腈使得乙腈的浓度为15％，然后用0.45μm微孔滤膜过滤。
纯化条件与梯度：
制备柱：DAC HB-50，富士硅胶
Figure PCTCN2017099882-appb-000003
-10μm-C18
检测波长：220nm，流速：50ml/min
流动相：A 0.1M NaH2PO4溶液，B 50％乙腈/A，0-60min：30％B-50％B
收集纯度99％以上的收集液，25℃浓缩，PS聚合物脱盐，加乙酸，浓缩，冻干，得缩宫素10.6g，HPLC图谱见附图18，ESI-MS图见附图19(oxytocin m/z calculated.1006.4；found1007.5[M+H]+)；以欧洲药典标准品作为标准物质，按欧洲药典缩宫素含量测定法，除去水分和醋酸，含量为98.2％，效价为588IU/mg。
实施例10
用方案1合成片段3：
称Boc-Leu-OH(1eq.,100mmol,23.1g)和BOP(1.01eq.,101mmol,44.7g)，并用用DCM200ml和DMF 20ml溶解，加DIPEA(1.2eq.,120mmol,21ml)，5min后，称H-Gly-NH2.HCl(1.1eq.,110mmol,12.2g)用DMF 100ml溶解，加TEA(1.1eq.,110mmol,15ml)，倒入以上反应液中，TLC(DCM:MeOH:AcOH＝100:20:1)跟踪监测,反应完全后，用0.5M HCl溶液析出白色稠状物质，EA溶解，萃取，水洗涤3遍，饱和氯化钠洗涤1遍，无水MgSO4干燥30min，过滤，浓缩至干，纯度99.1％，HPLC分析图谱见附图20。
将上述产物加200ml 30％TFA/DCM(三氟乙酸/二氯甲烷)溶液，反应30min，浓缩至干，真空干燥得产物H-Leu-Gly-NH2。纯度96％，产物25.6g，产率85％。HPLC分析图谱见附图21。

Claims (9)

  1. 一种多肽液相合成缩宫素的方法，其特征在于，包括如下步骤：
    (1)合成片段1：Boc-Cys(Acm)-Tyr(tBu)-OH；
    (2)合成片段2：H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe；
    (3)合成片段3：H-Leu-Gly-NH2；
    (4)合成化合物4：Boc-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe；
    (5)合成化合物5：Boc-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OH；
    (6)合成化合物6：
    Boc-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-Leu-Gly-NH2；
    (7)合成化合物7：
    Figure PCTCN2017099882-appb-100001
    (8)将化合物7用三氟乙酸进行脱帽反应，浓缩，得到缩宫素粗品溶液；
    (9)粗品用C18反相硅胶色谱纯化，制备得到缩宫素纯品。
  2. 根据权利要求1所述的一种多肽液相合成缩宫素的方法，其特征在于，所述步骤(1)中的片段1为：Boc-Cys(Acm)-Tyr(tBu)-OH；合成片段1的氨基酸单元为Boc-Cys(Acm)-OH和H-Tyr(tBu)-OH；缩合剂为DIC或DCC，活化剂为HOSu或HOBt；溶剂为THF和水，THF和水的比例为1:1—5:1，其中，所述溶剂还可以为DMF或DMSO。
  3. 根据权利要求1所述的一种多肽液相合成缩宫素的方法，其特征在于，所述步骤(2)中的片段2为：H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe；合成片段2包括如下步骤：
    (1)合成化合物2.1：Fmoc-Cys(Acm)-Pro-OMe：
    Fmoc-Cys(Acm)-OH与H-Pro-OMe.HCl的物质的量之比为1:1.05-1:1.5；缩合剂为DIC或DCC，缩合剂与H-Pro-OMe.HCl的物质的量之比为1:1；活化剂为HOSu或HOBt或HOOBt，活化剂与H-Pro-OMe.HCl的物质的量之比为1:1；碱为三乙胺(TEA)、二异丙基乙胺(DIPEA)或N-甲基吗啉(NMM)，碱与H-Pro-OMe.HCl的物质的量之比为1:1；溶剂为DMF、DMSO、DCM或THF；
    (2)合成化合物2.2：H-Cys(Acm)-Pro-OMe：
    化合物2.1的脱帽反应，脱帽试剂为二乙胺(DEA)或5％哌嗪/DCM；脱帽完成后，浓缩，用石油醚析出产物，过滤，真空干燥得到化合物2.2；
    (3)合成化合物2.3：Fmoc-Asn(Trt)-Cys(Acm)-Pro-OMe：
    化合物2.2与Fmoc-Asn(Trt)-OH的物质的量之比为1:1.05-1:1.5；缩合剂为DIC或DCC，缩合剂与Fmoc-Asn(Trt)-OH的物质的量之比为1:1；活化剂为HOSu、HOOBt或HOBt，活化剂与Fmoc-Asn(Trt)-OH的物质的量之比为1:1；溶剂为DMF、DMSO、DCM或THF；
    (4)合成化合物2.4：H-Asn(Trt)-Cys(Acm)-Pro-OMe：
    化合物2.3的脱帽反应，脱帽试剂为二乙胺(DEA)、5％哌嗪/DMF；脱帽完成后，浓缩，饱和NaHCO3溶液析出，过滤，水洗至中性，真空干燥，然后石油醚搅拌洗涤，过滤，真空干燥得化合物2.4；
    (5)合成化合物2.5：Fmoc-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe：
    化合物2.4与Fmoc-Gln(Trt)-OH的物质的量之比为1:1.05-1:1.5；缩合剂为DIC或DCC，缩 合剂与Fmoc-Gln(Trt)-OH的物质的量之比为1:1；活化剂为HOSu、HOOBt或HOBt，活化剂与Fmoc-Gln(Trt)-OH的物质的量之比为1:1；溶剂为DMF、DMSO、DCM或THF；
    (6)合成化合物2.6：H-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe：
    化合物2.5的脱帽反应，脱帽试剂为二乙胺(DEA)或5％哌嗪/DMF；脱帽完成后，浓缩，饱和NaHCO3溶液析出，过滤，水洗至中性，真空干燥，然后石油醚搅拌洗涤，过滤，真空干燥得化合物2.6；
    (7)合成化合物2.7：Fmoc-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe：
    化合物2.6与Fmoc-Ile-OH的物质的量之比为1:1.05-1:1.5；缩合剂为DIC或DCC，缩合剂与Fmoc-Ile-OH的物质的量之比为1:1；活化剂为HOSu、HOOBt或HOBt，活化剂与Fmoc-Ile-OH的物质的量之比为1:1；溶剂为DMF、DMSO、DCM或THF；
    (8)合成片段2：H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe：
    化合物2.7的脱帽反应，脱帽试剂为二乙胺(DEA)或5％哌嗪/DMF；脱帽完成后，浓缩，饱和NaHCO3溶液析出，过滤，水洗至中性，真空干燥，然后石油醚搅拌洗涤，过滤，真空干燥得片段2。
  4. 根据权利要求1所述的一种多肽液相合成缩宫素的方法，其特征在于，所述步骤(3)中的片段3为：H-Leu-Gly-NH2；合成片段3可以选择方案1或者方案2进行：
    方案1：氨基酸单元为Boc-Leu-OH、H-Gly-NH2.HCl；缩合剂为BOP、HBTU、TBTU、DIC或DCC/HOBt或HOSu；碱为TEA、NMM或DIPEA；溶剂为DMF、DMSO、THF或DCM；合成Boc-Leu-Gly-NH2后，用TFA脱去Boc得到H-Leu-Gly-NH2；
    方案2：氨基酸单元为Fmoc-Leu-OH、H-Gly-NH2.HCl；缩合剂为BOP、HBTU、TBTU、DIC或DCC/HOBt或HOSu；碱为TEA、NMM或DIPEA；溶剂为DMF、DMSO、THF或DCM；合成Fmoc-Leu-Gly-NH2后，脱去Fmoc保护基得到H-Leu-Gly-NH2；脱帽试剂为二乙胺(DEA)或5％哌嗪/DCM。
  5. 根据权利要求1所述的一种多肽液相合成缩宫素的方法，其特征在于，所述步骤(4)中的化合物4为：Boc-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe；
    合成化合物4包括如下步骤：片段1与片段2的物质的量之比为1.05-1.5:1；缩合剂为BOP、DCC、DIC或叠氮试剂，缩合剂与片段1的物质的量之比为1:1；活化剂为HOSu或HOBt，活化剂与片段1的物质的量之比为1:1；溶剂为DCM、THF、DMF或DMSO。
  6. 根据权利要求1所述的一种多肽液相合成缩宫素的方法，其特征在于，所述步骤(5)中的化合物5为：Boc-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OH；
    合成化合物5包括如下步骤：皂化试剂为LiOH或NaOH，其物质的量为化合物4的5-15当量，浓度为0.05-0.2M；溶剂为THF、DMF、甲醇、乙腈或DMSO。
  7. 根据权利要求1所述的一种多肽液相合成缩宫素的方法，其特征在于，所述步骤(6)中的化合物6为：
    Boc-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-Leu-Gly-NH2；
    合成化合物6包括如下步骤：化合物5与片段3的物质的量之比为：1:1.05-2；
    缩合剂为DCC、DIC、BOP或叠氮试剂；活化剂为HOSu、HOOBt或HOBt；溶剂为THF、DMF或DMSO。
  8. 根据权利要求1所述的一种多肽液相合成缩宫素的方法，其特征在于，所述步骤(7)： 合成化合物7，所用试剂为碘，其物质的量为化合物6的5-10当量，浓度为0.1-0.5M；溶剂为DMF；反应完成后，用0.1％硫代硫酸钠溶液析出固体，水洗，真空干燥得到化合物7。
  9. 根据权利要求8所述的一种多肽液相合成缩宫素的方法，其特征在于：所述化合物7的氨基酸序列为：
    Figure PCTCN2017099882-appb-100002
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