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Solid-phase synthesis method of Cilengitide

A cilengitide and solid-phase synthesis technology, which is applied in the field of solid-phase cyclization technology to synthesize cyclic peptides, can solve problems such as unfavorable industrial production, reduced cyclization yield, troublesome purification, etc., and achieves low cost, simple post-processing, The effect of short reaction time

Active Publication Date: 2012-10-17
YANCHENG KAILI PHARMA
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Problems solved by technology

[0005] At present, the preparation methods of cyclic peptides mainly include liquid phase synthesis process, solid phase synthesis precursor peptide cyclization process in liquid phase, and solid phase synthesis process. The first two synthesis processes are the synthesis of precursor peptides in liquid phase. This method requires the reactants to be reacted in a very dilute solvent (10 -3 ~10 -4 mol / L), and intermolecular reactions easily occur to form linear or cyclic polymers, which greatly reduces the cyclization yield, brings troubles to subsequent purification, and produces a large amount of waste liquid in large-scale production. conducive to industrial production

Method used

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  • Solid-phase synthesis method of Cilengitide
  • Solid-phase synthesis method of Cilengitide

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Preparation of Fmoc-L-Asp(OtBu)-Wang Resin

[0028] Weigh 10g of Wang Resin with a substitution degree of 0.5mmol / g into the reactor, add an appropriate amount of DCM, swell for 30min, and remove the DCM; dissolve 6.17g of Fmoc-L-Asp-OtBu, 2.40ml of DIC, and 2.1g of HOBT In 30ml of DMF, activate at 0-5°C for 15min, add the activation solution into the reactor containing Wang Resin, react for 10min, then add 0.18g of DMAP, react at 0-30°C for 1-5h. After the reaction, add 1ml of acetic anhydride and 0.5ml of pyridine to block the unreacted hydroxyl reagent of Wang Resin, and after 1 hour of blocking reaction, wash with 80ml of DMF, DCM, CH 3 Wash with OH and DMF 2, 1, 1, 2 times, 1 min each time. After testing, Fmoc-L-Asp(OtBu)-Wang Resin with a substitution degree of 0.47mmol / g was obtained.

Embodiment 2

[0030] Cilengitide precursor:

[0031] Preparation of A-Wang Resin (Fmoc-D-Phe-N-Me-L-Val-L-Arg(Mtr)-Gly-L-Asp(OtBu)-Wang Resin)

[0032] Add Fmoc-L-Asp(OtBu)-Wang Resin to the reactor, swell with DMF for 30min, remove the solvent, add 80ml 25% piperidine-DMF to react for 5min, wash with 80ml DMF once (3min), add 80ml 25% piperidine-DMF reaction for 15min; wash with 80ml DMF, DCM, CH 3 Wash 2, 1, 1, 2 times with OH and DMF, 1 min each time; dissolve 4.45g Fmoc-Gly-OH, 5.68g HBTU, 2.03g HOBt in 30ml of DMF, add 2.45ml of DIEA after dissolution, 0 Activate at ~5°C for 15 minutes, add the activation solution into the above reactor, react at 0-30°C for 1-3 hours, and detect the end point of the reaction with the ninhydrin method. Using the above method to sequentially couple Fmoc-L-Arg(Mtr)-OH, Fmoc-N-Me-L-Val, Fmoc-D-Phe-OH, finally get Fmoc-D-Phe-N-Me-L-Val -L-Arg(Mtr)-Gly-L-Asp(OtBu)-Wang Resin.

Embodiment 3

[0034] Precursor peptide of cilengitide: preparation of B-Wang Resin (D-Phe-N-Me-L-Val-L-Arg(Mtr)-Gly-L-Asp-Wang Resin)

[0035] Be the Fmoc deprotecting agent of Fmoc-D-Phe-N-Me-L-Val-L-Arg(Mtr)-Gly-L-Asp(OtBu)-Wang Resin with volume ratio of 25% piperidine-DMF, Add 25% piperidine-DMF 80ml for the first time, react for 5min, wash with 80ml DMF once (3min), add 25% piperidine-DMF 80ml for the second time, react for 15min, wash with 80ml DMF, DCM in turn 、CH 3 Wash 2, 1, 1, 2 times with OH and DMF, each time for 1 min. After washing, D-Phe-N-Me-L-Val-L-Arg(Mtr)-Gly-L-Asp(OtBu)-Wang Resin.

[0036] Add 80% PhOH-DCM solution with a volume ratio of 100ml and a catalytic amount of TFA to remove OtBu, react for 8h; wash with 80ml of DMF, DCM, CH 3 Wash 2, 1, 1, 2 times with OH and DMF, 1 min each time, to obtain D-Phe-N-Me-L-Val-L-Arg(Mtr)-Gly-L-Asp-Wang Resin.

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Abstract

The invention discloses a solid-phase synthesis method of Cilengitide. The method consists of: taking Fmoc-amino acid and Wang Resin as raw materials, using DMF or DCM as a solvent, adopting an organic base as an activating reagent, employing piperidine-DMF as an Fmoc protecting group removal reagent, and using solid-phase stepwise synthesis method to synthesize a Cilengitide precursor peptide A-Wang Resin; respectively removing Fmoc and OtBu from A-Wang Resin so as to obtain a Cilengitide precursor peptide B-Wang Resin; adding the organic base for solid-phase cyclization, and cracking resin to obtain a Cilengitide crude peptide; and carrying out purification and freeze-drying, thus obtaining Cilengitide. Employing a solid-phase cyclization technology, the method of the invention has the advantages of simple synthesis operation, high yield, easy purification, small environmental pollution, as well as low cost, and has the characteristics of objective economic value and broad application value.

Description

technical field [0001] The invention relates to a method for synthesizing cyclic peptides by solid-phase cyclization technology, in particular to designing a synthetic route of cilengitide. Background technique [0002] Cilengitide (Cilengitide), Chinese nickname: ring (L-arginylglycyl-L-aspartyl-D-phenylalanyl-N-methyl-L-valyl), is a A new class of synthetic anticancer drugs. Merkel's research found that cilengitide combined with chemoradiotherapy (combined and adjuvant temozolomide plus radiotherapy) may prolong survival. At the same time, as the first integrin inhibitor anti-tumor drug, it has entered phase III clinical trials. The important mechanism is Targets tumors and blood supply structures that provide nutrients to tumors and promote cancer cell growth by blocking blood vessel growth. [0003] The molecular formula of cilengitide is: C 27 h 40 N 8 o 7 , with the following structure: [0004] [0005] At present, the preparation methods of cyclic peptides ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/64C07K1/06C07K1/04
CPCY02P20/55
Inventor 杨毅跃姚程成康国伟鲁尧
Owner YANCHENG KAILI PHARMA
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