WO2017101810A1 - 一种利那洛肽的合成方法 - Google Patents

一种利那洛肽的合成方法 Download PDF

Info

Publication number
WO2017101810A1
WO2017101810A1 PCT/CN2016/110106 CN2016110106W WO2017101810A1 WO 2017101810 A1 WO2017101810 A1 WO 2017101810A1 CN 2016110106 W CN2016110106 W CN 2016110106W WO 2017101810 A1 WO2017101810 A1 WO 2017101810A1
Authority
WO
WIPO (PCT)
Prior art keywords
fmoc
linaclotide
resin
cys
tbu
Prior art date
Application number
PCT/CN2016/110106
Other languages
English (en)
French (fr)
Inventor
陈学明
吴靖康
宓鹏程
陶安进
袁建成
Original Assignee
深圳翰宇药业股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳翰宇药业股份有限公司 filed Critical 深圳翰宇药业股份有限公司
Priority to US16/062,079 priority Critical patent/US10442838B2/en
Priority to EP16874867.1A priority patent/EP3392266B1/en
Priority to ES16874867T priority patent/ES2812798T3/es
Publication of WO2017101810A1 publication Critical patent/WO2017101810A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • 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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • C07K1/067—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups for sulfur-containing functions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
    • 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 technical field of drug synthesis, in particular to a method for synthesizing linaclotide.
  • Linaclotide is a GC-C (intestinal epithelial cell uridine cyclase C) receptor agonist.
  • IBS-C intestinal epithelial cell uridine cyclase C
  • the drug was developed by Ironwood Pharmaceuticals Co., Ltd.
  • the compound has a structure of 14 amino acids containing three pairs of disulfide bonds. It can be obtained by cell expression and chemical synthesis. Its structure is as follows:
  • Method (1) using Trt as the side chain protecting group of Cys, the synthesis of linear peptide is completed by Fmoc solid phase peptide synthesis method, and the crude peptide is obtained, and the target peptide is obtained by one-step oxidation by liquid phase oxidation;
  • method (2) Using Trt and Acm as Cys side chain protecting groups, Fmoc solid phase peptide synthesis method to obtain partially protected linear peptides, and then using semi-selective strategy to complete the synthesis of disulfide bonds;
  • Method (3) using [2Mmt+2Acm+ respectively Three completely selective strategies for the synthesis of linaclotide by 2Trt], [2Acm+2Trt+2pMeOBzl], [2StBu+2Trt+2pMeOBzl].
  • the method (2) is a semi-selective localization oxidation method in which a pair of disulfide bonds are oxidized in position relative to the method (1), and the number of formation of different isomers is reduced, but the isomers are also unavoidable. produce.
  • the paper also It is directly mentioned that the formation of two pairs of disulfide bonds by iodine oxidation will seriously reduce the yield of crude peptides.
  • the investigator chose to form three pairs of disulfide bonds using three different complete selectivity, but none of the target products were obtained.
  • Chinese patents CN 104231051A, CN 104628826A, CN 104163853A, CN 104844693A, CN 102875655A describe a method for forming a three-pair disulfide bond by one-step oxidation: firstly synthesizing linaclotide resin, and then cleavage of linaclotide resin to remove all protecting groups. And the resin solid phase carrier to obtain the linaclotide linear crude peptide, and finally the linear peptide is subjected to a one-step oxidation reaction using an oxidation system.
  • CN 104231051A, CN 104163853A, CN 102875655A adopt GHS/GSSH oxidation system
  • CN 104844693A is oxidized by cysteine hydrochloride/DMSO buffer solution. system.
  • the one-step oxidation method can convert the linear peptide to the target structure through the buffer system as much as possible, the generation of disulfide mismatch isomer impurities is still unavoidable, and the yield is low.
  • the present invention is directed to the problem of producing isomer impurities due to mismatching of disulfide bonds in the above synthesis method, resulting in low purity and yield of the product, and providing a synthesis of three groups by completely selective formation of three pairs of disulfide bonds.
  • the method of lophin thereby achieving the directed and efficient synthesis of three pairs of fully crossed disulfide bonds.
  • the raw materials used in the process of the invention are relatively low in cost, in particular in the formation of a third pair of disulfide bonds, the use of less expensive methyl protected cysteine, and simultaneous demethylation and formation of a third p-disulfide
  • the key is simple in operation, low in cost and economical and practical.
  • a method for preparing linaclotide comprises the following steps:
  • linaclotide precursor resin Fmoc-Tyr(tBu)-OH is reacted with a carrier resin to obtain Fmoc-Tyr(tBu)-resin; Fmoc-Tyr(tBu)-resin is used as a solid phase carrier, one by one
  • the coupling method is coupled to Fmoc-AA-OH in the order of C-terminus to N-terminus to obtain linaclotide precursor resin, wherein the Cys side chain corresponding to the 5th and 13th positions of linaclotide is protected by Me, corresponding to Lina
  • the Cys side chain of position 1 and 6 of loperopeptide is protected by Mmt, and the Cys side chain corresponding to the 2nd and 10th positions of linaclotide is protected by Dpm;
  • the carrier resin described in the step 1) is a wang resin or a 2-chloro resin, and the degree of substitution of the resin is 0.1-1.0 mmol/g, preferably 0.2-0.8 mmol/g. More preferably, it is 0.2-0.5 mmol/g.
  • the Fmoc-AA-OH coupled in the order of C-terminus to N-terminus in the step 1) is Fmoc-Cys(Me)-OH, Fmoc-Gly-OH.
  • Methods of obtaining a linaclotide precursor resin include:
  • the detection method to be applied is any method known in the art for achieving this purpose, such as chromatography or chemical calibration, and it is preferred to use an agent which can determine the end point of the reaction, preferably ninhydrin.
  • an agent which can determine the end point of the reaction preferably ninhydrin.
  • ninhydrin if the resin develops color, it means that there is a free amine in the polypeptide, that is, there is no protecting group on the amine.
  • the reagent for removing Fmoc in the step b) is a 20% piperidine/DMF solution (DBLK), that is, a mixed solution of piperidine:DMF (volume ratio) of 1:4.
  • DBLK 20% piperidine/DMF solution
  • the coupling agent in step c) is a composition of DIC and Compound A or a combination of DIPEA and Compound A and Compound B, wherein Compound A is HOBt or HOAt, and Compound B is PyBOP, PyAOP, HATU, HBTU Or TBTU, preferably a combination of DIC and Compound A.
  • DIPEA: A: B 2.0: 1.1: 1.0.
  • the reaction of step c) is carried out in a solid phase reaction column.
  • the solid phase reaction column is not particularly limited and may be any solid phase reaction column that can achieve this.
  • the time for the coupling reaction of each amino acid is usually 1.5 to 4 hours, preferably 2 to 3 hours;
  • the pressure is preferably atmospheric pressure, and may be carried out under a suitably increased or decreased pressure;
  • the temperature is preferably room temperature (ie, 20 ⁇ 5 ° C) can also be carried out at a suitably elevated or lowered temperature.
  • the resin is swollen prior to each coupling step
  • the solvent used in the swelling step can be any agent in the art that can achieve this purpose, such as DMF, NMP, dichloromethane, etc., preferably DMF.
  • the solvent used in the washing step may be any reagent in the art that can achieve the purpose, such as DMF, NMP, dichloromethane, etc., preferably DMF.
  • the deprotecting agent described in the step 2) is a mixed solution of TFA/DCM, and the volume concentration of the TFA in the mixed solution is 1% to 10%, preferably 1% to 5%. %, the end point of the reaction is that the solution changes from red to colorless.
  • the oxidizing agent described in the step 3) is selected from the group consisting of H 2 O 2 , NCS, preferably NCS; the solvent used is selected from the group consisting of DMF, NMP, dichloromethane, preferably DMF.
  • step 4) in the lysate is TFA, H 2 O, PhOMe, mixtures of different proportions of thioanisole, preferably TFA, H 2 O, PhOMe, The thioanisole was mixed in a volume ratio of 90:5:4:1.
  • the oxidizing agent described in the step 5) is selected from the group consisting of H 2 O 2 and NCS, and the molar ratio of the oxidizing agent to the monodithiocyclic peptide obtained in the step 4) is 1:10- 10:1;
  • the oxidizing agent is NCS in an amount of from 1:1 to 10:1, preferably 2:1, in molar ratio to the monodithiocyclic peptide obtained in step 4).
  • the solvent used is selected from the group consisting of methanol, ethanol, acetone, tetrahydrofuran, acetonitrile, and a mixed solution of the above solvents in water at different ratios. Preference is given to a mixture of acetonitrile and water in a volume ratio of acetonitrile to water of from 1:1 to 1:5, preferably 1:1.
  • the reagent for synchronizing demethylation and oxidation used in the step 6) is selected from the group consisting of horseradish peroxidase, mushroom tyrosinase, and monoamine oxidase to demethylate and
  • the mass ratio of the reagent realized by oxidation synchronously to the bisdithiocyclic peptide obtained in the step 5) is 0.5:1000-10:100; preferably the horseradish peroxidase is used in an amount of the mass of the bisdithiocyclic peptide obtained in the step 5).
  • the ratio is from 0.5:100 to 10:100, more preferably from 1.5:100 to 2.5:100, most preferably 2.0:100.
  • the reversed-phase high-pressure liquid chromatography comprises: using a reverse phase octadecylsilane as a stationary phase, a 0.1% by volume aqueous acetic acid solution/acetonitrile as a mobile phase, and a mobile phase of a volume ratio of 0.1% by volume aqueous acetic acid solution to acetonitrile. It is preferably from 98:2 to 50:50, more preferably from 80:20 to 60:40, most preferably 70:30.
  • the peak fraction of interest was collected and concentrated to freeze.
  • the oxidizing agent used to form the first pair of disulfide bonds and the second pair of disulfide bonds may be the same (for example, NCS)
  • the protecting group Mmt of the first and sixth Cys is in a diluted TFA solution.
  • the protecting group Dpm of the 2nd and 10th Cys must be removed in the concentrated TFA, so that the first pair is selectively formed.
  • the strategy of disulfide bond and second pair of disulfide bond is to first remove the Mmt protecting group from the mixture of linaclotide precursor resin in TFA/DCM, and then solid phase oxidize to form the first pair of disulfide.
  • the bond is then cleavage of the resin in the linaclotide precursor resin containing the monodisulfide ring.
  • the lysate also removes the protecting group Dpm and is then liquid phase oxidized to form a second pair of disulfide bonds.
  • the present invention adopts a method for synthesizing linaclotide by completely selectively forming three pairs of disulfide bonds: first solid phase synthesis of linaclotide precursor resin, followed by solid phase oxidation to form a first a pair of disulfide bonds, after the cleavage reaction, liquid phase oxidation again to form a second pair of disulfide bonds, and finally the methyl group of the methyl protected cysteine is removed, and the third pair of disulfide bonds are oxidatively coupled to obtain a benefit Nunopeptide.
  • the method of the present invention avoids the production of disulfide bond mismatches by completely selectively forming three pairs of disulfide bonds to obtain higher purity and yield; meanwhile, the occurrence of linaclotide precursor resin occurs.
  • the solid phase oxidizes the first pair of disulfide bonds to reduce the difficulty of forming the first pair of disulfide bonds; and the raw material used in the preparation of the third pair of disulfide bonds is a cheaper methyl protected cystease Amino acid, and the removal of the methyl group of the cysteine protecting group and the oxidative coupling are simultaneously achieved.
  • the synthesis method has the advantages of high product purity, high yield, simple and easy raw materials, low cost, simple process and stable process, and is suitable for large-scale production, and has wide application prospects in the field of polypeptide drug synthesis technology.
  • Figure 1 is a schematic diagram of a synthetic route of the present invention.
  • the raw materials and reagents used in the preparation of linaclotide are commercially available and purchased from Jill Biochemical (Shanghai) Co., Ltd., Chengdu Zhengyuan Biochemical Technology Co., Ltd. and Suzhou Tianma Fine Chemicals Co., Ltd.
  • the reaction was carried out at room temperature for 2 hours, and the reaction end point was detected by ninhydrin (if the resin was colorless and transparent, the reaction was terminated; if the resin developed color, the reaction was prolonged for 1 hour).
  • the resin was washed 3 times with DMF (100 ml/time), deprotected twice by DBLK (100 ml/time) for 6 min, 8 min, DMF washed resin 6 times (100 ml/time), and the ninhydrin detection resin was colour.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

一种采用完全选择性形成三对二硫键合成利那洛肽的方法，其包括1)固相合成利那洛肽前体树脂；2)固相氧化形成第一对二硫键；3)液相氧化形成第二对二硫键4)脱除甲基保护的半胱氨酸的甲基，同时氧化偶联第三对二硫键得到利那诺肽。该反应条件温和、成本低、收率高、产物纯度高、工艺简单且工艺稳定、适用于规模化生产。

Description

一种利那洛肽的合成方法 技术领域
本发明涉及药物合成技术领域，具体涉及一种利那洛肽的合成方法。
背景技术
利那洛肽是一种GC-C(肠上皮细胞尿苷酸环化酶C)受体激动剂，2012年8月美国FDA批准用于治疗成人慢性特发性便秘和便秘型肠易激综合症(IBS-C)。该药物由Ironwood制药公司开发，化合物结构为14个氨基酸组成的含有三对二硫键的多肽，可以通过细胞表达和化学合成制得，其结构如下：
Figure PCTCN2016110106-appb-000001
Benitez等人2010年在Peptide Science上进行了相关的报道。文献中采用三种方法对利那洛肽进行尝试性的合成。方法(1)：采用Trt为Cys的侧链保护基团，用Fmoc固相肽合成方法完成线性肽的合成，得到粗肽后用液相氧化的方法一步氧化得到目标肽；方法(2)：采用Trt和Acm为Cys侧链保护基团，Fmoc固相肽合成方法得到部分保护的线性肽，然后用半选择的策略完成二硫键的合成；方法(3)：分别采用[2Mmt+2Acm+2Trt]、[2Acm+2Trt+2pMeOBzl]、[2StBu+2Trt+2pMeOBzl]三种完全选择性策略合成利那洛肽。
方法(1)中，虽然合成步骤简单，只使用了一种Cys的保护基团。但是，对于需要定位形成三对二硫键的肽来说，随机氧化会得到许多种不同的二硫键错配异构体，尽管可以通过一些缓冲溶液体系来使得氧化过程中尽可能大的转变为目标分子，但是始终无法避免其他异构体的产生。这种操作容易使得目标肽粗肽纯化偏低，收率不高，很难进行大规模生产。同时，采用该方法进行氧化，对外界条件比如温度等的依赖性也非常高，在不同的环境下，自然氧化所得到的产品收率相差也非常大，不利于产品质量的控制。方法(2)为一种半选择性定位氧化的方法，相对于方法(1)，定位氧化了一对二硫键，降低了形成不同异构体形成的数量，但是同样无法避免异构体的产生。同时，论文中也 直接提到，采用碘氧化形成两对二硫键，粗肽收率将严重下降。方法(3)中，研究者选择采用三种不同的完全选择性形成三对二硫键，但均未得到目标产物。
目前，针对利那洛肽还未有比较高效的制备方法。中国专利CN 104231051A、CN 104628826A、CN 104163853A、CN 104844693A、CN 102875655A介绍了一步氧化形成三对二硫键的方法：首先合成利那洛肽树脂，然后将利那洛肽树脂裂解脱除所有保护基和树脂固相载体得到利那洛肽线性粗肽，最后对线性肽采用氧化体系进行一步氧化反应。其中CN 104231051A、CN 104163853A、CN 102875655A采用GHS/GSSH氧化体系；CN 104628826A采用单质碘在pH＝6-13的磷酸钠缓冲溶液中氧化；CN 104844693A采用半胱氨酸盐酸盐/DMSO缓冲溶液氧化体系。虽然一步氧化法能够通过缓冲体系使线性肽尽量向目标结构转化，但仍然无法避免二硫键错配异构体杂质的产生，收率较低。
由此可见，亟需探索一种反应条件温和、成本低、收率高、产物纯度高、工艺简单且工艺稳定、适用于规模化生产的利那洛肽合成方法。
发明内容
本发明针对上述合成方法中存在的由于二硫键错配而产生异构体杂质，致使产物的纯度和收率偏低的问题，提供一种采用完全选择性形成三对二硫键合成利那洛肽的方法，从而实现定向高效合成三对全交叉的二硫键。
本发明方法中所使用的原料成本较低，特别是在形成第三对二硫键过程中采用较便宜的甲基保护的半胱氨酸，并且一步同时脱甲基和形成第三对二硫键，操作简单、成本低，具有经济实用的价值。
为实现上述目的，本发明提供的技术方案如下：
一种利那洛肽的制备方法，包括如下步骤：
1)制备利那洛肽前体树脂：Fmoc-Tyr(tBu)-OH和载体树脂反应，获得Fmoc-Tyr(tBu)-树脂；以Fmoc-Tyr(tBu)-树脂为固相载体，采用逐一偶联的方式按照C端到N端的顺序偶联Fmoc-AA-OH，获得利那洛肽前体树脂，其中对应利那洛肽第5、13位的Cys侧链采用Me保护，对应利那洛肽第1、6位的Cys侧链采用Mmt保护，对应利那洛肽第2、10位的Cys侧链采用Dpm保护；
2)用脱保护剂脱除步骤1)所得的利那洛肽前体树脂的Mmt保护基团；
3)用氧化剂氧化步骤2)所得的利那洛肽前体树脂，形成第一对二硫键，得到含单二硫环的利那洛肽前体树脂；
4)用裂解液切割步骤3)所得的含单二硫环的利那洛肽前体树脂中的树脂，同时脱除Dpm保护基团，得到单二硫环肽；
5)用氧化剂氧化步骤4)所得的单二硫环肽，形成第二对二硫键，得到双二硫环肽；
6)脱除步骤5)所得的双二硫环肽中Cys的甲基保护基团，同时氧化形成第三对二硫键，得到利那洛肽。
在本发明的制备利那洛肽的方法中，步骤1)中所述的载体树脂为wang树脂或2-氯树脂，树脂替代度为0.1-1.0mmol/g，优选0.2-0.8mmol/g，更优选0.2-0.5mmol/g。
在本发明的制备利那洛肽的方法中，步骤1)中所述的按照C端到N端的顺序偶联Fmoc-AA-OH依次为Fmoc-Cys(Me)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Dpm)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Mmt)-OH、Fmoc-Cys(Me)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(tBu)-OH、Fmoc-Cys(Dpm)-OH、Fmoc-Cys(Mmt)-OH。获得利那洛肽前体树脂的方法包括：
a)Fmoc-Tyr(tBu)-OH和载体树脂反应，获得Fmoc-Tyr(tBu)-树脂；
b)脱除Fmoc，接着用溶剂洗涤树脂，直至用检测方法检测到完全脱除Fmoc为止；
c)将合适量的待偶联氨基酸和偶联剂在溶剂中溶解并活化后，一起加入到固相反应柱中，直至用检测方法检测到反应终止为止；
d)重复b)和c)；
其中，应用的检测方法是本领域已知的可实现此目的的任意方法，例如色谱法或化学标定法，优选使用可判定反应终点的试剂，优选茚三酮。当使用茚三酮时，若树脂显色则说明多肽中有游离的胺，即胺上无保护基。
其中，在步骤b)中脱除Fmoc的试剂为20％的哌啶/DMF溶液(DBLK)，即哌啶：DMF(体积比)为1:4的混合溶液。
其中，在步骤c)中所述偶联剂为DIC和化合物A的组合物或DIPEA和化合物A和化合物B的组合物，其中化合物A为HOBt或HOAt，化合物B为PyBOP、PyAOP、HATU、HBTU或TBTU，优选为DIC和化合物A的组合物。进一步地，偶联剂中各成分的比例以摩尔比计为DIC:A＝1.2:1.1，DIPEA:A:B＝2.0:1.1:1.0。
其中，步骤c)的反应在固相反应柱中进行。对固相反应柱无特别限制，可为可实现此目的的任意固相反应柱。此外，每种氨基酸进行偶联反应的时间通常为1.5-4小时，优选2-3小时；压力优选为常压，也可在适当提高或降低的压力下进行；温度优选为室温(即20±5℃)，也可在适当提高或降低的温度下进行。
其中，将树脂在各偶联步骤之前进行溶胀，所述溶胀步骤中所使用的溶剂可以为本领域中能实现该目的的任何试剂，例如DMF、NMP、二氯甲烷等，优选DMF。
其中，所述洗涤步骤中所使用的溶剂可以为本领域中能实现该目的的任何试剂，例如DMF、NMP、二氯甲烷等，优选DMF。
在本发明的制备利那洛肽的方法中，步骤2)中所述的脱保护剂为TFA/DCM的混合液，混合液中TFA的体积浓度为1％-10％，优选1％-5％，反应终点为溶液由红色变为无色。
在本发明的制备利那洛肽的方法中，步骤3)中所述的氧化剂选自H2O2、NCS，优选NCS；所用溶剂选自DMF、NMP、二氯甲烷，优选DMF。
在本发明的制备利那洛肽的方法中，步骤4)中所述的裂解液为TFA、H2O、PhOMe、苯甲硫醚的不同比例的混合物，优选TFA、H2O、PhOMe、苯甲硫醚按照体积比为90:5:4:1混合。
在本发明的制备利那洛肽的方法中，步骤5)中所述的氧化剂选自H2O2、NCS，氧化剂与步骤4)所得的单二硫环肽的摩尔比为1:10-10:1；优选氧化剂为NCS，其用量为与步骤4)所得的单二硫环肽的摩尔比为1:1-10:1，优选2:1。所用溶剂选自甲醇、乙醇、丙酮、四氢呋喃、乙腈以及上述溶剂与水的不同比例的混合溶液。优选乙腈与水的混合液，乙腈与水的体积比例为1:1-1:5，优选1:1。
在本发明的制备利那洛肽的方法中，步骤6)中所用的使脱甲基和氧化同步实现的试剂选自辣根过氧化酶、蘑菇酪氨酸酶、单胺氧化酶，使脱甲基和氧化同步实现的试剂与步骤5)所得的双二硫环肽的质量比例为0.5:1000-10:100；优选辣根过氧化酶，其用量与步骤5)所得的双二硫环肽的质量比例为0.5:100-10:100，更优选1.5:100-2.5:100，最优选2.0:100。
在本发明的制备利那洛肽的方法中，采用反相高压液相色谱法进行纯化。双二硫环肽和最终产物利那洛肽均可采用反相高压液相色谱法进行纯化。进一步地，所述反相高压液相色谱法包括：以反相十八烷基硅烷为固定相，以0.1体积％醋酸水溶液/乙腈为流动相，流动相0.1体积％醋酸水溶液与乙腈的体积比例优选为98:2至50:50，更优选80:20至60:40，最优选70:30。收集目的峰馏分，浓缩冻干。
在本发明中，尽管形成第一对二硫键和第二对二硫键所使用的氧化剂可以相同(例如NCS)，但是由于第1位、第6位Cys的保护基团Mmt在稀TFA溶液中脱除，而第2位、第10位Cys的保护基团Dpm则必须在浓TFA中脱除，因此为实现选择性地形成第一对 二硫键和第二对二硫键，所采取的策略为首先使利那洛肽前体树脂在TFA/DCM的混合液中脱除Mmt保护基团，再固相氧化形成第一对二硫键；随后在裂解含单二硫环的利那洛肽前体树脂中的树脂同时，裂解液也使保护基团Dpm脱除，再液相氧化形成第二对二硫键。
在本发明中，采用Fmoc-Cys(Me)-OH作为原料，有利于最后脱除半胱氨酸的保护基团甲基的同时氧化偶联第三对二硫键得到利那洛肽。该步骤中优选辣根过氧化酶，其既用作脱甲基试剂，也用作氧化剂，从而达到脱甲基和氧化同步实现的目的。
综上，与现有技术相比，本发明采用完全选择性地形成三对二硫键而合成利那洛肽的方法：先固相合成利那洛肽前体树脂，然后固相氧化形成第一对二硫键，在进行裂解反应之后再次液相氧化形成第二对二硫键，最后脱除甲基保护的半胱氨酸的甲基，同时氧化偶联第三对二硫键得到利那诺肽。
本发明的方法通过完全选择性地形成三对二硫键，避免了二硫键错配异构体的产生，以得到较高的纯度和收率；同时，在利那洛肽前体树脂发生裂解反应之前，固相氧化第一对二硫键，以降低第一对二硫键形成的难度；并且在第三对二硫键的制备中采用的原料为较便宜的甲基保护的半胱氨酸，而且使半胱氨酸的保护基团甲基的脱除和氧化偶联同步实现。
该合成方法具有产物纯度高、收率高、原料简单易得、成本低、工艺简单且工艺稳定，适用于规模化生产等优点，同时在多肽药物合成技术领域具有广泛的应用前景。
附图说明
图1为本发明的合成路线方案图。
具体实施方式
下面通过实施例对本发明作进一步的详细说明，旨在用于说明本发明而非限定本发明。应当指出，对于本技术人员而言，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也同样落入本发明的保护范围之内。
本发明所使用的缩写的含义列于下表中。
Figure PCTCN2016110106-appb-000002
Figure PCTCN2016110106-appb-000003
Figure PCTCN2016110106-appb-000004
利那洛肽的制备方法中所用原料和试剂均可由市场购得，分别购于吉尔生化(上海)有限公司、成都郑源生化科技有限公司和苏州天马精细化学品股份有限公司。
实施例1：替代度为0.50mmol Fmoc-Tyr(tBu)-Wang树脂的制备
称取替代度为1.0mmol/g的Wang树脂100g于固相反应柱中，加入150ml DMF，氮气鼓泡溶胀60分钟；称取Fmoc-Tyr(tBu)-OH(45.9g，100mmol)、HOBt(16.2g，120mmol)、DMAP(1.2g，10mmol)，用100ml DMF溶解，0℃下加入DIC(20.3ml，117.1mmol)，活化5分钟，加入反应柱。反应两小时后，加入醋酸酐(70ml)和(60ml)吡啶，混合封闭24小时，DCM洗涤3次(100ml/次)。用甲醇收缩树脂，抽干得到Fmoc-Tyr(tBu)-Wang树脂150g，检测替代度为0.50mmol/g。
实施例2：利那洛肽前体树脂的制备
称取实施例1制备的替代度为0.50mmol/g的Fmoc-Tyr(tBu)-Wang树脂50g(25mmol)于固相反应柱中，加入50ml DMF，氮气鼓泡溶胀60分钟；然后用DBLK脱保护2次(50ml/次)，分别为6min、8min，DMF洗涤6次(100ml/次)。称取Fmoc-Cys(Me)-OH(48.7g，75mmol)和HOBt(11.7g，75mmol)用100ml DMF溶解，冰水浴下加入DIC(13ml，75mmol)活化3min后，将混合液加入到反应柱中，室温反应2小时，以茚三酮检测反应终点(如树脂无色透明则终止反应；如树脂显色则延长反应1小时)。反应结束，用DMF洗涤树脂3次(100ml/次)，加入DBLK脱保护2次(100ml/次)，分别为6min、8min，DMF洗涤树脂6次(100ml/次)，茚三酮检测树脂有颜色。
重复上述偶联操作，按照从C端到N端的肽序依次偶联Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Dpm)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Mmt)-OH、Fmoc-Cys(Me)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(tBu)-OH、Fmoc-Cys(Dpm)-OH、Fmoc-Cys(Mmt)-OH。在各偶联步骤中，加入替代度为0.50mmol/g由上一步所得的树脂50g，上述各氨基酸、HOBt、DIC 75mmol。在以上偶联操作全部结束后，用甲醇收缩树脂，抽干，得到利那洛肽前体树脂107.6g。
实施例3：脱除保护基Mmt
将实施例2得到的利那洛肽前体树脂107.6g，用1升DMF溶液溶胀1小时，减压抽掉溶液，用DCM洗涤两次(500ml/次)。树脂用2％TFA/DCM(v/v)的溶液250ml洗涤，每次2分钟，直到树脂颜色由红色变为无色，然后用DCM洗涤2次(500ml/次)，DMF洗涤2次(500ml/次)，减压抽掉溶液。
实施例4：含单二硫环的利那洛肽前体树脂的制备
向实施例3得到的脱除保护基Mmt的树脂中加入1升DMF，然后加入NCS(5.34g，40mmol)，反应半小时后，减压抽掉溶液，用DMF洗涤3次(500ml/次)，加入甲醇500ml收缩30分钟，抽掉甲醇，真空干燥得树脂92.2g。
实施例5：单二硫环肽的制备
将实施例4得到的树脂92.2g加入到1L三口瓶中，加入预先配置好的TFA:H2O:PhOMe:苯甲硫醚＝90:5:4:1(V:V)900ml，室温反应2小时，减压过滤树脂，收集滤液。用少量TFA洗涤树脂，合并滤液。将滤液缓慢加入10L冰乙醚中沉淀，离心，冰乙醚洗涤5次(5L/次)，减压干燥得到粗肽25.3g，HPLC纯度70.6％。
实施例6：双二硫环肽的制备
将实施例4得到的16.1g单二硫环肽溶于50％乙腈/水(v/v)的溶液500ml中，加入NCS(2.67mg，0.02mmol)，室温反应2小时，氧化完毕后，将该混合物直接上样10cm×25cm制备柱纯化制备。纯化条件：以反相十八烷基硅烷为固定相；流动A相为0.1％醋酸/水(v/v)的溶液，B相为乙腈，A：B＝70:30(体积比)等梯度洗脱；流速：70-80ml/min；检测波长：230nm；收集目的峰馏分，浓缩冻干，得纯品14.5g，纯度96％，收率90％。
实施例7：利那洛肽的制备
将实施例5得到的14.5g双二硫环肽溶解于300ml乙腈中，加入磷酸二氢钠缓冲溶液280ml(pH＝6)，然后加入辣根过氧化酶300mg，反应1小时后，将该混合物直接上样10cm×25cm制备柱纯化制备。以反相十八烷基硅烷为固定相；流动A相为0.1％醋酸/水(v/v)的溶液，B相为乙腈，A：B＝70:30(体积比)等梯度洗脱；流速：70-80ml/min；检测波长：280nm；收集目的峰馏分，浓缩冻干，得纯品10.0g，纯度为99.5％，收率70％。

Claims (10)

  1. 一种利那洛肽的合成方法，包括如下步骤：
    1)制备利那洛肽前体树脂：Fmoc-Tyr(tBu)-OH和载体树脂反应，获得Fmoc-Tyr(tBu)-树脂；以Fmoc-Tyr(tBu)-树脂为固相载体，采用逐一偶联的方式按照C端到N端的顺序偶联Fmoc-AA-OH，获得利那洛肽前体树脂，其中对应利那洛肽第5、13位的Cys侧链采用Me保护，对应利那洛肽第1、6位的Cys侧链采用Mmt保护，对应利那洛肽第2、10位的Cys侧链采用Dpm保护；
    2)用脱保护剂脱除步骤1)所得的利那洛肽前体树脂的Mmt保护基团；
    3)用氧化剂氧化步骤2)所得的利那洛肽前体树脂，形成第一对二硫键，得到含单二硫环的利那洛肽前体树脂；
    4)用裂解液切割步骤3)所得的含单二硫环的利那洛肽前体树脂中的树脂，同时脱除Dpm保护基团，得到单二硫环肽；
    5)用氧化剂氧化步骤4)所得的单二硫环肽，形成第二对二硫键，得到双二硫环肽；
    6)脱除步骤5)所得的双二硫环肽中Cys的甲基保护基团，同时氧化形成第三对二硫键，得到利那洛肽。
  2. 根据权利要求1所述的利那洛肽的合成方法，其中，步骤1)中所述的按照C端到N端的顺序偶联Fmoc-AA-OH为Fmoc-Cys(Me)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Dpm)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Mmt)-OH、Fmoc-Cys(Me)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(tBu)-OH、Fmoc-Cys(Dpm)-OH、Fmoc-Cys(Mmt)-OH。
  3. 根据权利要求1所述的合成方法，其中，步骤1)制备利那洛肽前体树脂按照以下方式进行：a)Fmoc-Tyr(tBu)-OH和载体树脂反应，获得Fmoc-Tyr(tBu)-树脂；b)脱除Fmoc，接着用溶剂洗涤树脂，直至用检测方法检测到完全脱除Fmoc为止；c)将合适量的待偶联氨基酸和偶联剂在溶剂中溶解并活化后，一起加入到固相反应柱中，直至用检测方法检测到反应终止为止；d)重复b)和c)，
    其中，脱除Fmoc的试剂为20％的哌啶/DMF溶液(DBLK)，即哌啶：DMF(体积比)为1:4的混合溶液；
    其中，偶联剂为DIC和化合物A的组合物或DIPEA和化合物A和化合物B的组合物， 其中化合物A为HOBt或HOAt，化合物B为PyBOP、PyAOP、HATU、HBTU或TBTU，优选为DIC和化合物A的组合物；偶联剂中各成分的比例以摩尔比计为DIC:A＝1.2:1.1，DIPEA:A:B＝2.0:1.1:1.0；
    其中，将树脂在偶联之前进行溶胀，所使用的试剂选自DMF、NMP、二氯甲烷。
  4. 根据权利要求1所述的合成方法，其中，步骤1)中所述的载体树脂为wang树脂或2-氯树脂，树脂替代度为0.1-1.0mmol/g，优选0.2-0.8mmol/g，更优选0.2-0.5mmol/g。
  5. 根据权利要求1-4中任一项所述合成方法，其中，步骤2)中所述脱保护剂为TFA/DCM的混合液，混合液中TFA的体积浓度为1％-10％，优选1％-5％。
  6. 根据权利要求1-4中任一项所述的合成方法，其中，步骤3)中所述氧化剂选自H2O2、NCS；所用溶剂选自DMF、NMP、二氯甲烷。
  7. 根据权利要求1-4中任一项所述的合成方法，其中，步骤4)中所述裂解液为TFA、H2O、PhOMe、苯甲硫醚的混合物，体积比TFA:H2O:PhOMe:苯甲硫醚＝90:5:4:1。
  8. 根据权利要求1-4中任一项所述的合成方法，其中，步骤5)中所述氧化剂选自H2O2、NCS，氧化剂与步骤4)所得的单二硫环肽的摩尔比为1:10-10:1；优选氧化剂为NCS，其用量为与步骤4)所得的单二硫环肽的摩尔比为1:1-10:1，优选2:1；所用溶剂选自甲醇、乙醇、丙酮、四氢呋喃、乙腈、以及上述溶剂与水的不同比例的混合溶液；优选乙腈与水的混合液，乙腈与水的体积比为1:1-1:5，优选1:1。
  9. 根据权利要求1-4中任一项所述的合成方法，其中，步骤6)中，所用的脱甲基和氧化试剂选自辣根过氧化酶、蘑菇酪氨酸酶、单胺氧化酶，脱甲基和氧化试剂与步骤5)所得的双二硫环肽的质量比例为0.5:1000-10:100；优选辣根过氧化酶，其用量与步骤5)所得的双二硫环肽的质量比例为0.5:100-10:100，更优选1.5:100-2.5:100，最优选2.0:100。
  10. 根据权利要求1-4中任一项所述的合成方法，其中还包括步骤：7)采用反相高压液相色谱法纯化利那洛肽。
PCT/CN2016/110106 2015-12-18 2016-12-15 一种利那洛肽的合成方法 WO2017101810A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US16/062,079 US10442838B2 (en) 2015-12-18 2016-12-15 Linaclotide synthesis method
EP16874867.1A EP3392266B1 (en) 2015-12-18 2016-12-15 Linaclotide synthesis method
ES16874867T ES2812798T3 (es) 2015-12-18 2016-12-15 Método de síntesis de linaclotida

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510964573.0 2015-12-18
CN201510964573.0A CN106892968B (zh) 2015-12-18 2015-12-18 一种利那洛肽的合成方法

Publications (1)

Publication Number Publication Date
WO2017101810A1 true WO2017101810A1 (zh) 2017-06-22

Family

ID=59055764

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/110106 WO2017101810A1 (zh) 2015-12-18 2016-12-15 一种利那洛肽的合成方法

Country Status (5)

Country Link
US (1) US10442838B2 (zh)
EP (1) EP3392266B1 (zh)
CN (1) CN106892968B (zh)
ES (1) ES2812798T3 (zh)
WO (1) WO2017101810A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111100188A (zh) * 2018-10-25 2020-05-05 成都医学院 一种制备蜘蛛抗菌肽的方法
EP4194464A1 (en) 2021-12-13 2023-06-14 Chemi SPA Manufacturing process for the production of linaclotide

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107383171A (zh) * 2017-08-15 2017-11-24 苏州科技大学 一种通过二次环化固相合成普卡那肽的方法
CN109311941B (zh) * 2017-12-14 2021-09-07 深圳市健元医药科技有限公司 一种合成利那洛肽的方法
CN110498841B (zh) * 2018-05-16 2021-07-06 深圳翰宇药业股份有限公司 一种含丙氨酸的首尾环肽合成方法
CN111057129B (zh) * 2018-10-16 2024-01-16 深圳翰宇药业股份有限公司 一种用于合成含有两对二硫键的多肽的制备方法及其试剂盒，以及普利卡那肽的制备方法
CN111499693B (zh) * 2020-04-27 2023-05-12 汉肽生物医药集团有限公司 一种固液结合制备利那洛肽的方法
CN111732632B (zh) * 2020-07-16 2021-12-21 台州吉诺生物科技有限公司 一种利那洛肽的合成方法
CN113480634A (zh) * 2021-08-09 2021-10-08 甘肃瑞德林生物有限公司 一种兰瑞肽的制备方法
CN113754735B (zh) * 2021-10-11 2023-07-21 杭州信海医药科技有限公司 一种利那洛肽的制备方法
CN113956333B (zh) * 2021-12-22 2022-03-29 浙江湃肽生物有限公司南京分公司 一种利那洛肽的合成和纯化方法
CN115894630A (zh) * 2022-12-05 2023-04-04 四川轻化工大学 一种利那洛肽的制备方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102875655A (zh) * 2012-09-29 2013-01-16 深圳翰宇药业股份有限公司 一种合成利那洛肽的方法
CN103626849A (zh) * 2013-11-27 2014-03-12 深圳翰宇药业股份有限公司 一种利那洛肽的制备方法
CN104163853A (zh) * 2014-07-25 2014-11-26 杭州诺泰制药技术有限公司 一种制备利那洛肽的方法
CN104231051A (zh) * 2013-06-06 2014-12-24 深圳翰宇药业股份有限公司 一种利那洛肽的制备方法
CN104844693A (zh) * 2015-06-10 2015-08-19 成都圣诺生物科技股份有限公司 一种合成利那洛肽的方法
CN104974229A (zh) * 2015-07-06 2015-10-14 泰州施美康多肽药物技术有限公司 一种利那洛肽的固相合成方法
CN105017387A (zh) * 2015-07-31 2015-11-04 济南康和医药科技有限公司 一种制备利那洛肽的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015022575A2 (en) * 2013-08-13 2015-02-19 Auro Peptides Ltd A process for the preparation of gc-c agonist

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102875655A (zh) * 2012-09-29 2013-01-16 深圳翰宇药业股份有限公司 一种合成利那洛肽的方法
CN104231051A (zh) * 2013-06-06 2014-12-24 深圳翰宇药业股份有限公司 一种利那洛肽的制备方法
CN103626849A (zh) * 2013-11-27 2014-03-12 深圳翰宇药业股份有限公司 一种利那洛肽的制备方法
CN104163853A (zh) * 2014-07-25 2014-11-26 杭州诺泰制药技术有限公司 一种制备利那洛肽的方法
CN104844693A (zh) * 2015-06-10 2015-08-19 成都圣诺生物科技股份有限公司 一种合成利那洛肽的方法
CN104974229A (zh) * 2015-07-06 2015-10-14 泰州施美康多肽药物技术有限公司 一种利那洛肽的固相合成方法
CN105017387A (zh) * 2015-07-31 2015-11-04 济南康和医药科技有限公司 一种制备利那洛肽的方法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111100188A (zh) * 2018-10-25 2020-05-05 成都医学院 一种制备蜘蛛抗菌肽的方法
EP4194464A1 (en) 2021-12-13 2023-06-14 Chemi SPA Manufacturing process for the production of linaclotide
WO2023110781A1 (en) 2021-12-13 2023-06-22 Chemi Spa Manufacturing process for the production of linaclotide

Also Published As

Publication number Publication date
US10442838B2 (en) 2019-10-15
ES2812798T3 (es) 2021-03-18
EP3392266B1 (en) 2020-05-27
CN106892968B (zh) 2021-11-05
EP3392266A9 (en) 2019-04-10
CN106892968A (zh) 2017-06-27
EP3392266A1 (en) 2018-10-24
EP3392266A4 (en) 2019-08-14
US20180371022A1 (en) 2018-12-27

Similar Documents

Publication Publication Date Title
WO2017101810A1 (zh) 一种利那洛肽的合成方法
CN102875655B (zh) 一种合成利那洛肽的方法
WO2017097194A1 (zh) 一种全固相制备卡贝缩宫素的方法
CN104974237B (zh) 一种片段法固相合成齐考诺肽的方法
CN106167514A (zh) 一种利那洛肽的合成和纯化方法
WO2018205401A1 (zh) 一种普利卡那肽的制备方法
WO2012083861A1 (zh) 一种制备醋酸阿托西班的方法
CN102702320A (zh) 一种制备爱啡肽的方法
CN106554391B (zh) 一种海洋生物肽Xen2174的合成方法
CN106854235B (zh) 一种固相片段法合成卡贝缩宫素
CN103992389B (zh) 一种固环合成去氨加压素的方法
CN106478805A (zh) 一种glp-1衍生物的制备方法
CN103467573B (zh) 一种卡贝缩宫素的制备方法
CN109053863A (zh) 一种低成本制备高纯度利那洛肽的方法
CN112585153A (zh) 一种化合物或其盐及其制备方法与应用
WO2020077781A1 (zh) 一种用于合成含有两对二硫键的多肽的制备方法及其试剂盒，以及普利卡那肽的制备方法
WO2019019492A1 (zh) 一种合成pt141的方法
CN110642936B (zh) 一种制备特立帕肽的方法
CN106008674A (zh) 一种制备利那洛肽的方法
CN112876541B (zh) 一种地加瑞克的固相合成方法
CN112175067B (zh) 一种替度鲁肽的制备方法
CN103275207A (zh) 一种制备奈西立肽的方法
CN113801199A (zh) 一种卡贝缩宫素的全固相合成方法
CN109748950B (zh) 一种固相合成血管升压素受体肽激动剂selepressin的方法
CN111732632A (zh) 一种利那洛肽的合成方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16874867

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2016874867

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2016874867

Country of ref document: EP

Effective date: 20180718