WO2020125045A1 - Procédé de synthèse de romidepsine - Google Patents

Procédé de synthèse de romidepsine Download PDF

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Publication number
WO2020125045A1
WO2020125045A1 PCT/CN2019/101597 CN2019101597W WO2020125045A1 WO 2020125045 A1 WO2020125045 A1 WO 2020125045A1 CN 2019101597 W CN2019101597 W CN 2019101597W WO 2020125045 A1 WO2020125045 A1 WO 2020125045A1
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WO
WIPO (PCT)
Prior art keywords
fmoc
phase
romidepsin
synthesis method
coupling agent
Prior art date
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Ceased
Application number
PCT/CN2019/101597
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English (en)
Chinese (zh)
Inventor
陈学明
林欣媛
宓鹏程
陶安进
袁建成
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Hybio Pharmaceutical Co Ltd
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Hybio Pharmaceutical Co Ltd
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Publication of WO2020125045A1 publication Critical patent/WO2020125045A1/fr
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    • 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
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • 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 solid-phase synthesis of medicinal chemistry, in particular to a method for synthesizing romidepsin.
  • Romidepsin whose English name is Romidepsin, and its chemical name is (1S, 4S, 7Z, 10S, 16E, 21R)-7-ethylidene-4,21-diisopropyl-2-oxa-12, 13-disulfide-5,8,20,23-tetraazabicyclo[8,7,6]cosadec-16-ene-3,6,9,19,22-pentanone with molecular formula C 24 H 36 N 4 O 6 S 2 is a bicyclic tetrapeptide with a stable hydrophobic structure. The unique disulfide bond in its structure is a key group for its activity.
  • FDA US Food and Drug Administration
  • CTCL cutaneous T-cell lymphoma
  • Romidepsin is a histone deacetylase (HDACs) inhibitor, enters the cytoplasm through the tumor cell membrane, and the intracellular disulfide bond is reduced by glutathione to a sulfhydryl group, which is combined with zinc in zinc-dependent HDACs. Play the role of inhibiting HDACs, thereby further inducing tumor cell differentiation and apoptosis.
  • HDACs histone deacetylase
  • valine Methyl ester is used as the raw material, coupled with the corresponding amino acid, then the methyl ester is removed by lithium hydroxide, and then the ester is formed under the conditions of DEAD and PPh 3 , and finally the disulfide bond is synthesized by iodine oxidation, after a total of 14 steps to synthesize romidi Xin.
  • this method is cumbersome and not simple enough. More importantly, the total liquid yield is only about 18%.
  • a preparation method based on solid-phase synthesis of romidepsin is disclosed: first, the resin is coupled to the carboxyl group on 3-hydroxy-7-mercapto-4-heptenoic acid, and then romidepsin The upper 4 amino acids are coupled in sequence, and then the hydroxyl group is removed, cyclized into a disulfide bond, and an amide bond to form romidepsin.
  • the synthetic route is as follows:
  • the invention includes the following steps:
  • the protective groups mentioned in the present invention are protective groups commonly used in the field of amino acid synthesis to protect the amino acid main chain and side chains such as amino groups, carboxyl groups, mercapto groups and other groups that interfere with synthesis. Reactions occur during the process to form impurities.
  • amino acids that need to protect the side chains in the present invention those skilled in the art are well aware of their side chain structure and know that common protecting groups are used to protect amino, carboxyl, mercapto and other groups on the amino acid side chains Group, wherein R is a thiol protecting group, and Fmoc is an N-terminal protecting group of amino acids.
  • Fmoc-L-Val-OH refers to the Fmoc protective group L-Val coupled to the N-terminus
  • Fmoc-L-Thr-OH refers to the L-Thr coupled Fmoc protective group at the N-terminus
  • Fmoc-D-Cys (R)-OH refers to the D-Cys coupled with the Fmoc protecting group at the N-terminus and the R protecting group at the side chain mercapto group
  • Fmoc-D-Val-OH refers to the Fmoc protecting group coupled to the N-terminus D-Val
  • the mercapto protecting group is trityl, 4-methoxytrityl or benzyl, more preferably trityl.
  • the solid phase synthetic resin is CTC Resin (CTC resin) or Wang Resin (king resin), more preferably CTC Resin, and most preferably CTC Resin with a substitution degree of 0.5 mmol/g.
  • the activator is a combination of one or more of DIPEA, diethylamine, triethylamine, morpholine, and N-methylmorpholine, preferably DIPEA.
  • step 1) of the preparation method of the present invention preferably, the molar ratio of the resin, the activator and Fmoc-L-Val-OH is 1:2-10:10-60, preferably 1:6:30.
  • step 1) is:
  • DIPEA is added to activate, and then coupled with the washed and swollen resin to obtain intermediate I.
  • the solvent used to dissolve, wash and swell the resin in this step is preferably DMF.
  • the polypeptide chain extension coupling means that after Fmoc-L-Val-OH is coupled with the resin, the remaining amino acids and 3-tert-butoxy-7-(R)- Mercapto-4-heptenoic acid undergoes a condensation reaction (condensation reaction of main chain amino group and carboxyl group) with the previous coupled amino acid one by one according to the order of its connection in the romidepsin structure.
  • a condensation reaction condensation reaction of main chain amino group and carboxyl group
  • DBLK is preferred for the present invention N-terminal protecting group.
  • step 2) is:
  • step 2) removes the Fmoc protecting group with DBLK for intermediate I, and then adds Fmoc-L-Thr-OH, coupling agent and DMAP for coupling. Then repeat the above operation to complete the coupling of Fmoc-D-Cys(R)-OH, Fmoc-D-Thr-OH, 3-tert-butoxy-7-(R)-mercapto-4-heptenoic acid one by one Intermediate V is obtained.
  • the coupling agent is preferably a HOBt/DIC dual system coupling agent, a PyBOP/HOBt dual system coupling agent or a TBTU/HOBt dual system coupling agent, and most preferably a PyBOP/HOBt dual system coupling agent.
  • the proportion of each component in the art is certain and well-known in the art, and will not be repeated here.
  • the solvent used for dissolution in step 2) is preferably one or two of DMF, DCM, NMP, and DMSO, and more preferably a mixed solvent with a volume ratio of DMF:NMP of 1:1.
  • the amount of the coupling agent used in step 2) is, on a molar basis, the coupling agent: the amount of solid phase synthetic resin used in step 1) is 2-4:1, preferably 3:1,
  • the amount of DMAP used in step 2) is on a molar basis.
  • the amount of DMAP: the amount of solid phase synthetic resin used in step 1) is 0.1-0.5:1, preferably 0.2:1.
  • Step 3) of the preparation method of the present invention is:
  • the reaction time is 1-5 hours, preferably 2 hours; the reaction temperature is preferably -5°C to 100°C, more preferably 30°C.
  • the solvent used for dissolution is preferably DMF, DCM or THF.
  • step 3 the amount of piperidine used is on a molar basis.
  • the amount of piperidine: solid phase synthetic resin used in step 1) is 1-3:1, preferably 2:1.
  • step 3 the dosage of the p-toluenesulfonyl chloride is on a molar basis, and the dosage ratio of the solid-phase synthetic resin in the step 1) is 1-2:1, preferably 1.5:1.
  • step 3 the amount of DMAP used is on a molar basis, and the ratio of the amount of solid phase synthetic resin used in DMAP: step 1) is 0.1-1:1, preferably 0.2:1.
  • step 3 the amount of DABCO used is on a molar basis.
  • the solid phase synthetic resin in DABCO: step 1) is 1-5:10, preferably 2:10.
  • step 4) of the preparation method of the present invention
  • the aqueous solution of the lysis reagent TFA is preferably a mixed lysis solution with a volume ratio of TFA:H 2 O of 95:5 and a volume ratio of TFA:EDT:PhOH:H 2 O of 95:5:3: 2.
  • Mixed lysate or volume ratio TFA:EDT:TIS:PhOH:H 2 O is 80:5:5:5:5.
  • step 5 of the preparation method of the present invention, after the intermediate VII is dissolved, an oxidizing agent is added to react to completion to obtain the intermediate VIII.
  • the reaction time is 1-10 hours, preferably 5 hours.
  • the reaction temperature is preferably -5°C to 100°C, more preferably 35°C.
  • the oxidant is hydrogen peroxide, horseradish peroxidase, DMSO, ferric chloride hexahydrate, air, ammonium persulfate, preferably ferric chloride hexahydrate.
  • the solvents used are methanol, ethanol, acetone, tetrahydrofuran, acetonitrile, and mixed solutions of the above solvents and water in different ratios; preferably, a mixed solution of acetonitrile and water.
  • step 6 after the intermediate VIII is dissolved, a coupling agent is added, the reaction is performed for 1-10 hours, and romidepsin is prepared by purification.
  • the reaction time is preferably 3 hours; the reaction temperature is preferably -5°C to 100°C, more preferably 35°C.
  • the coupling agent is preferably DIAD, PPh 3 and TsOH.
  • the solvent is methanol, ethanol, acetone, tetrahydrofuran, acetonitrile; preferably tetrahydrofuran.
  • the purification is purification by reverse-phase high-pressure liquid chromatography.
  • the reverse-phase high-pressure liquid chromatography includes: taking reverse-phase octadecylsilane as a stationary phase, and 0.2% aqueous acetic acid solution /Acetonitrile is the mobile phase, and the ratio of the mobile phase is preferably 98:2 to 50:50, more preferably 80:20 to 60:40, and most preferably 70:30.
  • the desired peak fractions were collected, concentrated and lyophilized.
  • the invention discloses a method for preparing romidepsin. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. In particular, it should be noted that all similar substitutions and modifications will be obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method of the present invention has been described through preferred embodiments, and it is obvious that relevant persons can modify or appropriately modify and combine the compounds and preparation methods described herein without departing from the content, spirit, and scope of the present invention. Apply the technology of the present invention.
  • all amino acids coupled with a protecting group are commercially available, and the protected amino acids in the present invention are purchased from Shanghai Jier Biochemical Co., Ltd.
  • the CTC Resin (CTC resin) and Wang Resin (Wang Resin) was purchased from Tianjin Nankai Hecheng Co., Ltd.
  • the method of the invention has simple steps and high synthesis efficiency, and can increase the total yield of romidepsin to about 40%.
  • the intermediate I was removed with 20% DBLK to remove the Fmoc protecting group, and then washed with DMF 6 times. A small amount of resin was taken for the ninhydrin test, and the resin developed color. Weigh 1.02g Fmoc-Thr-OH, 0.38g HOBt, 0.03g DMAP in a mixed solution of DMF and NMP with a volume ratio of 1:1, add 0.3mL DIC under ice water bath to activate, add it to a solid phase reaction column, and react at room temperature 2h (The end point of the reaction is determined by the ninhydrin method.
  • This oil was diluted 10 times with water using RP-HPLC system, wavelength 230nm, chromatographic column was 50 ⁇ 250mm reverse phase C18 column, 0.2% acetic acid aqueous solution/acetonitrile solution mobile phase was purified, the target peak fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain Romidepsin acetate refined peptide 0.25g, HPLC purity 98.5%, total yield 40.5%.

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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)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé de synthèse de romidepsine, comprenant les étapes consistant à : 1) coupler une résine synthétique en phase solide avec Fmoc-L-Val-OH sous l'action d'un activateur pour obtenir un intermédiaire I ; 2) selon une stratégie de synthèse en phase solide Fmoc, utiliser de manière séquentielle Fmoc-L-Thr-OH, Fmoc-D-Cys(R)-OH, Fmoc-D-Val-OH et de l'acide (R)-3-tert-butoxy-7-mercapto-4-hepténoïque d'une manière biunivoque pour réaliser un couplage d'extension de chaîne polypeptidique afin d'obtenir un intermédiaire V ; 3) retirer le groupe hydroxyle sur la chaîne latérale du résidu L-Thr de l'intermédiaire V pour former une double liaison, de façon à obtenir un intermédiaire VI ; 4) effectuer un craquage de l'intermédiaire VI et éliminer la résine et les groupes protecteurs de chaîne latérale, de manière à obtenir un intermédiaire VII ; 5) oxyder l'intermédiaire VII pour former une liaison disulfure intramoléculaire, de manière à obtenir un intermédiaire VIII ; et 6) soumettre l'intermédiaire VIII à une réaction d'estérification intramoléculaire pour obtenir de la romidepsine.
PCT/CN2019/101597 2018-12-19 2019-08-20 Procédé de synthèse de romidepsine Ceased WO2020125045A1 (fr)

Applications Claiming Priority (2)

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CN201811555690.1 2018-12-19
CN201811555690.1A CN111333697B (zh) 2018-12-19 2018-12-19 一种罗米地辛的合成方法

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CN115656372B (zh) * 2022-10-27 2024-10-11 北京斯利安药业有限公司 一种s-四氢叶酸异构体手性分析方法

Citations (3)

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CN102241736A (zh) * 2011-05-13 2011-11-16 江苏江神药物化学有限公司 抗肿瘤药物罗米迪司肽的关键中间体的合成方法
CN103897029A (zh) * 2012-12-27 2014-07-02 深圳翰宇药业股份有限公司 一种罗米地辛的制备方法
CN107778350A (zh) * 2016-08-25 2018-03-09 成都圣诺生物制药有限公司 一种合成罗米地辛的方法

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WO2016084100A2 (fr) * 2014-11-26 2016-06-02 Alaparthi Lakshmi Prasad Procédé nouveau et efficace de synthèse à grande échelle de romidepsine
WO2017068596A1 (fr) * 2015-10-20 2017-04-27 Msn Laboratories Private Limited Procédé amélioré pour la préparation de (1s, 4s, 7z, 10s, 16e, 21r)- 7-éthylidène-4,21-bis(1-méthyléthyl)-2-oxa-12,13-dithia-5, 8, 20, 23- tétraazabicyclo[8.7.6]tricos-16-ène-3, 6, 9, 19, 22-pentone

Patent Citations (3)

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CN102241736A (zh) * 2011-05-13 2011-11-16 江苏江神药物化学有限公司 抗肿瘤药物罗米迪司肽的关键中间体的合成方法
CN103897029A (zh) * 2012-12-27 2014-07-02 深圳翰宇药业股份有限公司 一种罗米地辛的制备方法
CN107778350A (zh) * 2016-08-25 2018-03-09 成都圣诺生物制药有限公司 一种合成罗米地辛的方法

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LI, K. W. ET AL.: "Total Synthesis of the Antitumor Depsipeptide FR-901, 228", J. AM. CHEM. SOC., vol. 118, no. 30, 31 December 1996 (1996-12-31), XP001019297 *
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CN111333697A (zh) 2020-06-26

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