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WO2020248360A1 - Procédé de synthèse de verakatide - Google Patents

Procédé de synthèse de verakatide Download PDF

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Publication number
WO2020248360A1
WO2020248360A1 PCT/CN2019/101609 CN2019101609W WO2020248360A1 WO 2020248360 A1 WO2020248360 A1 WO 2020248360A1 CN 2019101609 W CN2019101609 W CN 2019101609W WO 2020248360 A1 WO2020248360 A1 WO 2020248360A1
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WO
WIPO (PCT)
Prior art keywords
solid
phase
fmoc
resin
reaction
Prior art date
Application number
PCT/CN2019/101609
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English (en)
Chinese (zh)
Inventor
陈学明
陶志强
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.)
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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
Publication of WO2020248360A1 publication Critical patent/WO2020248360A1/fr

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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
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 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
    • C07K1/04—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
    • C07K1/06—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
    • 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 field of medicinal chemistry, in particular to a method for synthesizing verakapide.
  • Etelcalcetide is a calcimimetic developed by AMGEN INC. It is mainly used as a polypeptide drug for secondary hyperparathyroidism in hemodialysis treatment of adult patients with chronic kidney disease. It was launched in the United States on February 07, 2017 under the trade name Parsabiv. It is as good as cinacalcet in reducing the level of parathyroid hormone when used in secondary hyperparathyroidism, and because it can be administered intravenously after hemodialysis, it is better than the existing standard calcimimetic treatment The medicine cinacalcet.
  • the main chain of verakatide is composed of seven D-amino acids, and the side chain is connected to L-cysteine through a disulfide bond.
  • the peptide sequence is as follows:
  • the key to synthesizing verakapeptide by conventional methods is the construction of disulfide bonds.
  • the methods of constructing disulfide bonds in conventional peptide synthesis include air oxidation, iodine/acetic acid system oxidation, and hydrogen peroxide oxidation.
  • the specific selectivity of the reaction cannot be guaranteed, mismatched impurities will inevitably be produced, and the product purity is low, which increases the difficulty of purification.
  • Patent CN105504012 introduces a pseudo-dilution reaction to avoid the mismatch of disulfide bonds between polypeptide chains in the liquid phase reaction, but the solid phase disulfide bond reaction is a heterogeneous reaction, and the reaction conversion rate is low, resulting in the product Vera
  • the yield of carpeptide is less than 30%.
  • Patent CN106928320 and Patent CN201580029560.2 reported the synthesis of veracapeptide by 2,2-disulfide dipyridine activation method, but the equivalent amount of pyrithione compound is produced, which not only increases the difficulty of purification, but also causes pollution to the environment, which is not conducive to environmental protection. .
  • Patent CN106928321 introduces a method for all-liquid phase synthesis of verakapide, but the post-reaction treatment is complicated and the total yield is low.
  • the present invention uses Fmoc/tBu strategy to solid-phase connect main chain amino acid residues, and then solid-phase coupling side chain cysteine by electrochemical method. Finally, the precipitate is lysed to obtain verakatide. In this way, the problem of specific selectivity of the reaction can be avoided, the utilization of main chain fragments and the utilization of atoms can be improved, thereby reducing production costs, avoiding the use of 2,2-dithiodipyridine compounds, making it more environmentally friendly and in line with green chemistry idea.
  • the reaction process is as follows:
  • One aspect of the present invention provides a method for preparing verakatide, which comprises the following steps:
  • the solid-phase electrochemical oxidation method is: electrolyzing the polypeptide-solid-phase synthetic resin obtained in step 2) with the Cys side chain protection group removed in the electrolyte and the solution until the disulfide bond reaction is complete;
  • P is the side chain protecting group of Cys.
  • the electrochemical reaction temperature in step 3 is 25°C-65°C, preferably 40-60°C.
  • the electrolyte in step 3) is selected from tetrabutylammonium perchlorate, tetrabutylammonium tetrafluoroborate, tetrabutylammonium hexafluorophosphonium, preferably tetrabutyltetrafluoroborate Ammonium.
  • the platinum electrode is used for electrification and oxidation, and the current intensity is 5-20 mA; preferably 10-15 mA.
  • reaction time in step 3 is 2-100 hours.
  • the solid phase synthetic resin in step 1) is Rink Amide AM Resin, Rink Amide MBHA Resin, or Rink Amide Resin, etc.
  • the resin substitution degree is 0.1-1.0 mmol/g, preferably 0.2-0.8 mmol/g g, more preferably 0.3-0.5 mmol/g.
  • the preparation method of solid phase synthesis in step 1) refers to coupling Fmoc-AA-OH in the order of C-terminal to N-terminal, and the coupling order of Fmoc-AA-OH is sequential coupling of Fmoc-D -Arg(Pbf)-OH, Fmoc-D-Ala-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Ala-OH, Fmoc-Cys(P)-OH, then coupled with acetic acid.
  • P in Fmoc-Cys(P)-OH is Trt, Mmt or Dpm.
  • the solid-phase synthesis method includes: a) removing Fmoc, and then washing the resin with a solvent until the complete removal of Fmoc is detected by the detection method; b) the amino acid to be coupled (or After the acetic acid and the coupling agent are dissolved and activated in the solvent, they are added to the solid phase reaction column together until the termination of the reaction is detected by the detection method; c) repeat a) and b).
  • the reagent for removing Fmoc is a 20% piperidine/DMF solution (DBLK), that is, a piperidine:DMF (volume ratio) of 1:4 mixed solution.
  • DBLK 20% piperidine/DMF solution
  • the coupling agent in step a) is a combination of DIPCDI and compound A, wherein compound A is HOBt or HOAt, and compound B is PyBOP, PyAOP, HATU, HBTU or TBTU, preferably Composition of DIPCDI and Compound A.
  • the reaction of step 1) is carried out in a solid phase reaction column.
  • the solid-phase reaction column is not particularly limited, and can be any solid-phase reaction column that can achieve this purpose.
  • the coupling reaction time for each amino acid is usually 1.5-4 hours, preferably 2-3 hours; the pressure is preferably normal pressure, and can also be carried out under appropriately increased or reduced pressure; the temperature is preferably room temperature (ie 20 ⁇ 5°C), it can also be carried out at an appropriately increased or decreased temperature.
  • the reaction of step 1) preferably swells the solid-phase synthetic resin before coupling.
  • the washing and swelling steps can be carried out in the art using any reagent for achieving this purpose, including DMF, NMP, dichloromethane, etc., preferably DMF.
  • the detection method used in the reaction is any method known in the art that can achieve this purpose, such as chromatography or chemical calibration, preferably using a reagent that can determine the end point of the reaction, preferably ninhydrin, when ninhydrin is used , If the resin develops color, it indicates that there are free amines in the polypeptide, that is, there is no protecting group on the amine.
  • the selected coupling agent is DIPCDI+A or DIPEA+A+B, where A is HOBt or HOAt, and B is one of PyBOP, PyAOP, HATU, HBTU, and TBTU.
  • Step 2 The removing agent used is a mixture of TFA/DCM, the volume ratio of the solution is 1%-10%, preferably 1%-5%, and the reaction end point is that the solution turns from red to colorless.
  • Step 5 The purification step adopts reverse phase high pressure liquid chromatography.
  • the reverse-phase high-pressure liquid chromatography method includes: using reverse-phase octadecylsilane as a stationary phase, and using a 0.1% trifluoroacetic acid aqueous solution/acetonitrile as a mobile phase, collecting target peak fractions, and concentrating and lyophilizing.
  • D stands for D configuration amino acid
  • Mmt and Pbf are side chain protecting groups
  • the main chain is coupled through the ⁇ amino group and carboxyl group of the amino acid.
  • the main chain amino acid residues are solid-phase connected through the Fmoc/tBu strategy, and then the cysteine of the side chain is solid-phase oxidized by an electrochemical method, and then cleavage, precipitation, and purification are carried out to obtain verakapide.
  • This method can increase the utilization rate of fragmented peptide resin, increase the utilization rate of main chain fragments, and increase the utilization rate of atoms, thereby reducing production costs, avoiding the use of 2,2-dithiodipyridine compounds in the original patent, and being more environmentally friendly.
  • the concept of green chemistry is used to increase the utilization rate of fragmented peptide resin, increase the utilization rate of main chain fragments, and increase the utilization rate of atoms, thereby reducing production costs, avoiding the use of 2,2-dithiodipyridine compounds in the original patent, and being more environmentally friendly.
  • Example 2 250 mL of a 2% TFA/CH 2 Cl 2 solution was added to the peptide resin obtained in Example 1 to remove the Mmt protecting group. The reaction appeared red, react for 2 minutes, then aspirate the solution and repeat 10 times until the redness of the solution disappeared, wash with CH 2 Cl 2 3 times and then DMF 3 times.
  • the peptide resin obtained in Example 2 was added to a 1000ml three-necked flask, 500ml of DMF, 8.2g (25mmol) of tetrabutylammonium tetrafluoroborate, and 11.7g (75mmol) of cysteine hydrochloride monohydrate were added in sequence, Then, a platinum electrode (anode: 15mm ⁇ 15mm ⁇ 0.3mm, cathode: 15mm ⁇ 15mm ⁇ 0.3mm) was inserted, the current was controlled to 12mA, and the reaction was stirred at 40°C for 48 hours.
  • the energization was stopped, the filtration was performed, the DMF was washed 6 times, the DCM washed 3 times, the methanol was shrunk, and the peptide resin was vacuum dried to obtain 94.0 g of peptide resin, and the resin weight gain was 79.5%.
  • Example 4 The crude peptide obtained in Example 4 was purified by high performance liquid phase, using reverse-phase octadecylsilane as the stationary phase, and 0.1% trifluoroacetic acid aqueous solution/acetonitrile as the mobile phase. The target peak fractions were collected and concentrated and lyophilized. 16.8 g of refined peptide was obtained, with a purity of 99.98% and a yield of 64.5%.
  • Example 6 The original research method comparative example main chain peptide resin synthesis
  • Example 7 Comparative example of original research method, cracking and activation of 2,2-dithiodipyridine
  • Example 8 Comparative example of the original research method, oxidation to obtain crude peptide
  • Example 7 The activated crude peptide (10.7 g) in Example 7 was added to a 1.5% TFA aqueous solution (3.5 L), and L-Cys-OH (1.55 g, 1.1 eqv) was added and stirred for 2 hours. Detected by HPLC, the purity of the crude peptide is 40.31%.
  • Example 9 Comparative example of original research method, preparation of refined peptide by reversed-phase chromatography
  • Example 7 The crude peptide obtained in Example 7 was purified by HPLC to obtain 4.3 g of refined peptide with a purity of 98.7% and a yield of 32.4%.
  • the peptide resin obtained in Example 2 was added to a 1000ml three-necked flask, 500ml of DMF, 8.2g (25mmol) of tetrabutylammonium tetrafluoroborate, and 11.7g (75mmol) of cysteine hydrochloride monohydrate were added in sequence, Then, a platinum electrode (anode: 15mm ⁇ 15mm ⁇ 0.3mm, cathode: 15mm ⁇ 15mm ⁇ 0.3mm) was inserted, the current was controlled to 12mA, and the reaction was stirred at 60°C for 48 hours. After the reaction, power supply was stopped, filtration, DMF washing 6 times, DCM washing 3 times, methanol contraction, and vacuum drying to obtain 93.0 g of peptide resin, with a resin weight gain of 78.6%.
  • the peptide resin obtained in Example 2 was added to a 1000ml three-necked flask, 500ml DMF, 9.3g (25mmol) of tetrabutylammonium hexafluorophosphine, and 11.7g (75mmol) of cysteine hydrochloride monohydrate were added in sequence, Then, a platinum electrode (anode: 15mm ⁇ 15mm ⁇ 0.3mm, cathode: 15mm ⁇ 15mm ⁇ 0.3mm) was inserted, the current was controlled to 12mA, and the reaction was stirred at 40°C for 48 hours.
  • the power supply was stopped, the filter was filtered, the DMF was washed 6 times, the DCM washed 3 times, the methanol was shrunk, and the peptide resin was vacuum dried to obtain 94.1 g of peptide resin, and the resin weight gain was 79.5%.
  • the peptide resin obtained in Example 2 was added to a 1000ml three-necked flask, 500ml of DMF, 8.2g (25mmol) of tetrabutylammonium tetrafluoroborate, and 11.7g (75mmol) of cysteine hydrochloride monohydrate were added in sequence, Then, a platinum electrode (anode: 15mm ⁇ 15mm ⁇ 0.3mm, cathode: 15mm ⁇ 15mm ⁇ 0.3mm) was inserted, the current was controlled to 15mA, and the reaction was stirred at 40°C for 48 hours.
  • the energization was stopped, the filtration was performed, the DMF was washed 6 times, the DCM washed 3 times, the methanol was shrunk, and the peptide resin was vacuum dried to obtain 93.9 g of peptide resin, and the resin weight gain was 79.5%.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un procédé de préparation de verakatide, comprenant les étapes suivantes consistant à : 1) Préparer une résine synthétique en phase solide à chaîne principale de verakatide complètement protégée par synthèse en phase solide ; 2) réaliser un craquage pour éliminer un groupe protecteur P à chaîne latérale de Cys ; 3) effectuer une oxydation électrochimique en phase solide, coupler la cystéine par l'intermédiaire de liaisons disulfure ; 4) craquer tous les groupes protecteurs et la résine synthétique en phase solide pour obtenir un peptide brut de verakatide ; éventuellement, 5) effectuer une purification chromatographique pour obtenir un peptide fin de verakatide ; l'oxydation électrochimique en phase solide étant destinée à électrolyser la résine synthétique en phase solide du polypeptide obtenue à l'étape 2) avec le groupe protecteur à chaîne latérale de Cys éliminé dans l'électrolyte et mettre en solution jusqu'à ce que la réaction de liaison disulfure soit complète ; P représente le groupe protecteur à chaîne latérale de Cys. Le procédé selon l'invention peut augmenter le taux d'utilisation de la résine peptidique de fragment, augmenter le taux d'utilisation de fragments de chaîne principale et le taux d'utilisation d'atomes, ce qui permet de réduire les coûts de production, d'éviter l'utilisation de composés de 2,2-dithiodipyridine dans le brevet de recherche d'origine, qui est plus écologique et qui admet un concept de chimie verte.
PCT/CN2019/101609 2019-06-10 2019-08-20 Procédé de synthèse de verakatide WO2020248360A1 (fr)

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CN201910496387.7A CN112062811B (zh) 2019-06-10 2019-06-10 一种维拉卡肽的合成方法
CN201910496387.7 2019-06-10

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Publication number Priority date Publication date Assignee Title
CN112552376B (zh) * 2020-12-31 2022-11-25 江苏诺泰澳赛诺生物制药股份有限公司 一种纯化维拉卡肽的方法
CN114524860A (zh) * 2021-12-29 2022-05-24 深圳翰宇药业股份有限公司 一种Etelcalcetide的合成方法及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105504012A (zh) * 2014-09-30 2016-04-20 深圳翰宇药业股份有限公司 一种多肽的制备方法
CN106795201A (zh) * 2014-04-03 2017-05-31 美国安进公司 用于制备amg416的方法
CN107434820A (zh) * 2017-08-07 2017-12-05 南京工业大学 一种维拉卡肽的合成方法
US20190100554A1 (en) * 2017-10-03 2019-04-04 Chunghwa Chemical Synthesis & Biotech Co. Ltd. Method for synthesizing etelcalcetide or salts thereof
CN109734778A (zh) * 2019-03-07 2019-05-10 苏州科技大学 一种维拉卡肽的制备方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002329977A1 (en) * 2001-08-27 2003-03-10 Combimatrix Corporation In vitro protein translation microarray device
CN109280078B (zh) * 2018-10-30 2019-06-25 成都诺和晟泰生物科技有限公司 一种制备维拉卡肽的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106795201A (zh) * 2014-04-03 2017-05-31 美国安进公司 用于制备amg416的方法
CN105504012A (zh) * 2014-09-30 2016-04-20 深圳翰宇药业股份有限公司 一种多肽的制备方法
CN107434820A (zh) * 2017-08-07 2017-12-05 南京工业大学 一种维拉卡肽的合成方法
US20190100554A1 (en) * 2017-10-03 2019-04-04 Chunghwa Chemical Synthesis & Biotech Co. Ltd. Method for synthesizing etelcalcetide or salts thereof
CN109734778A (zh) * 2019-03-07 2019-05-10 苏州科技大学 一种维拉卡肽的制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ERAK, M. ET AL.: "Peptide Chemistry Toolbox–Transforming Natural Peptides into Peptide Therapeutics", BIOORGANIC & MEDICINAL CHEMISTRY, vol. 26, 31 January 2018 (2018-01-31), XP055765133, DOI: 20200226095214A *
HUANG, P. H. ET AL.: "Electro-Oxidative S-H/S-H Cross-Coupling with Hydrogen Evolution: Facile Access to Unsymmetrical Disulfides", ANGEW. CHEM. INT. ED., vol. 57, 29 May 2018 (2018-05-29), XP055763400, DOI: 20200226094816Y *

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CN112062811A (zh) 2020-12-11

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