CN104861042A - Method for preparing cetrorelix acetate through specific microwave synthesis - Google Patents
Method for preparing cetrorelix acetate through specific microwave synthesis Download PDFInfo
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Abstract
The invention belongs to the technical field of synthesis and preparation methods of polypeptide drugs, and particularly relates to a synthesis and preparation method for cetrorelix acetate. The method is unique and exclusive. Rink Amide-AM resin is taken as a carrier, a low-cost HBTU/DIEA (hexafluorophosphate/diisopropylethylamine) is taken as a condensing agent, and a specific microwave synthesis technology is adopted, so that the condensation efficiency is improved and the crude yield reaches 92%; obtained crude cetrorelix acetate is purified with a reversed-phase high-performance liquid chromatography (rHPLC) method and subjected to acetate transformation treatment by adopting strong anion exchange resin, the yield of obtained pure cetrorelix acetate reaches more than 40%, and the acetic acid content of cetrorelix acetate reaches international standard requirements. The method greatly shortens reaction time and increases final product yield, thereby having considerable economic and applicable values and wide application prospects.
Description
Technical field
The invention belongs to polypeptide drugs preparation method technical field, particularly a kind of method preparing cetrorelix acetate with specificity Microwave synthesize.
Background technology
Chinese: cetrorelix acetate
English name: cetrorelix Acetate
Structural formula: Ac-D-2-Nal-D-Cpa-D-3-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2
Molecular formula: C70H92N17O14Cl
Molecular weight: 1431.06
Cetrorelix acetate (cetrorelix Acetate) is a kind of effective Progesterone releasing hormone statin (LHRH) receptor antagonist, for controlling the hormesis of ovary, preventing the too early discharge of immature ovarian follicle, helping to become pregnant.Easily decomposed by the ferment in gi tract due to the own physical character (Mw value) of cetrorelixAcetate and destroy, not by orally using, therefore often make injection cetrorelix preparation, absorbed by subcutaneous or intramuscular injection, play a role rapidly.
Cetrorelix acetate is often called cetrorelix again, the luteotropic hormone (LH) of pituitary gland and the secretion of follicular stimulating hormone (FSH) is suppressed by dose-dependant, and can not affect progestin acceptor activity and by large quantity research, result display cetrorelix has good curative effect to ovarian cancer, prostate cancer, fibroma uteri, endometriosis and other diseases, and has prevention and improvement result to benign prostatauxe and ovarian hyperstimulation syndrome.
Cetrorelix preparation method in the market, have employed liquid phase synthesizing method more, all used in these methods toxicity larger as the reagent such as pyridine, ammoniacal liquor, if for a large amount of preparations of cetrorelix, surely understand this type of reagent of Long Term Contact, larger to person injury.The present invention utilizes Fmoc solid phase synthesis principle, develop the efficient synthetic technology of solid phase, adopt commonly to be easy to get, the source chemicals of low cost to be to synthesize preparation, by process optimization and distinctive microwave reaction technical finesse, cetrorelix crude yield is up to 92%, its sterling yield reaches 40%, substantially increases the yield of cetrorelix; The present invention also adopts strong anion exchange resin-acetic acid to turn salt technical finesse cetrorelix sterling, greatly reduces trifluoroacetic content in sterling, thoroughly removes the toxicity of trifluoracetic acid to organism; The present invention is simple to operate, and reaction process sampling is convenient, and be easy to middle control, and synthesis cycle is short, production cost is low, and by product is few, and product yield is high, is beneficial to scale operation rapidly.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method preparing cetrorelix acetate with specificity Microwave synthesize, and this preparation method's reaction conditions is gentle, reaction efficiency is high, cost is low, have the wide application prospect of producing cetrorelix acetate raw material.
The preparation method of cetrorelix acetate of the present invention, comprises solid phase polypeptide synthesis synthesis cetrorelix resin, obtains cetrorelix crude product after the sedimentation of cetrorelix resin cutting, cetrorelix purifying crude, turn salt freeze-drying after obtain cetrorelix acetate sterling; For achieving the above object, the present invention takes following technical scheme to prepare cetrorelix acetate:
(1) with Rink Resin for initial resin carrier, connect amino acid corresponding in cetrorelix product serial successively by solid-phase synthesis, obtain cetrorelix precursor peptide I-aminoresin as follows:
Fmoc-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-aminoresin
(2) take off D-Nal protecting group, carry out N and hold acetylization reaction, obtain cetrorelix precursor peptide II-aminoresin as follows:
Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-aminoresin
(3), after cutting, sedimentation, cetrorelix thick peptide III is obtained as follows:
Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2
(4) purifying, turn salt and freeze-drying obtains cetrorelix acetate IV.
In above-mentioned cetrorelix process for solid phase synthesis, step (1) described aminoresin is preferably Rink Amide-AM Resin.Be condensing agent and activating reagent with HBTU/DIEA in reaction, with 20%Pip/DMF solution for deprotecting regent, carry out the continuous condensating reaction of polypeptide.
Step (2) described acetylization reaction, acetylation reagent is volume ratio is aceticanhydride: the mixing solutions of DIEA: DMF=1: 1: 4 configurations, reaction solution volume is 1/3 ~ 1/2 of reactor volume.
Step (3) described cutting, precipitation reaction, the consumption of cutting reagent is 1g resin 8ml cutting reagent, and resin cutting twice, be blown into respectively in ice ether by cutting liquid, supernatant is removed in centrifugation, and repetitive operation obtains the thick product of polypeptide for 3 times.
Step (4) described purifying process, selects 5um C18 preparative column to carry out RPLC purifying, adopts acetic acid-strong anion exchange resin to turn salt technical finesse cetrorelix polypeptide, finally obtains cetrorelix acetate IV sterling.
Embodiment
To contribute to understanding the present invention by following embodiment, but content of the present invention can not be limited.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple replacements or deduction can also be made, all be considered as belonging to protection scope of the present invention.
Embodiment 1
1. aminoresin is swelling
Weigh the Rink-AM-Resin 0.15g that substitution value is 0.97 ~ 1.04mmol/g, be added to Peptide systhesis reactor from opening end, get DCM reagent and be added in reactor, resin is immersed in DCM solvent completely, fully contacts with solvent, swelling 2h.
The synthesis of 2.Fmoc-cetrorelix resin
Cetrorelix precursor peptide I-aminoresin is:
Fmoc-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-aminoresin
The protected amino acid that the present embodiment uses from resin start at the corresponding protected amino acid of 1st ~ 10 amino acid and molecular weight as shown in the table:
In the present invention, some conventional abbreviations have following implication:
Fmoc: fluorenylmethyloxycarbonyl 3-pyridyl-D-Ala:3-D-phenylalanine DMF:N, dinethylformamide
D-Cit:D-citrulline pbf:2,2,4,6,7-pentamethyl-Dihydrobenzofuranes-5-sulphonyl
D-2-Nal:D-2-naphthylalanine Pip: hexahydropyridine TFA: trifluoracetic acid tBu: the tertiary butyl
DIC:N, N-Diisopropylcarbodiimide DIEA:N, N-diisopropylethylamine HOBt:1-hydroxybenzotriazole
(1) activation method of protected amino acid
For the condensation of Fmoc-D-Ala-aminoresin; 22.36mgFmoc-D-Ala-OH and 20.74mg HBTU is taken in centrifuge tube according to the theoretical amino acid charging capacity calculated; add 2ml DMF to be dissolved; getting 10ulDIEA with liquid-transfering gun again joins in centrifuge tube, mixes the protected amino acid solution obtaining activating.
(2) washing methods of resin after deprotection
With vacuum pump, the deprotection solution in reactor is drained, then add the DMF solution of 1/3 ~ 1/2 reactor volume, the decolorization swinging table being placed in 30r/min shakes 1min, drain solution in reactor and add DMF washing again, repetitive operation 3 times.
After triketohydrindene hydrate color developing detection is qualified, the protected amino acid solution of above-mentioned activation is added in the reactor drained, then reactor is placed in the isothermal vibration device reaction 20 ~ 30min of 25 DEG C.Adopt above-mentioned same method, successively condensation 2nd ~ 10 Fmoc-protected amino acids, namely obtain cetrorelix precursor peptide I-aminoresin.
3. the crude yield of the thick peptide III of cetrorelix
Crude product after cutting sedimentation is latex state, and wherein containing more tfa salt and cutting reagent, the present invention adopts C18 chromatographic column to carry out desalination, to calculate crude product yield.
Sample dissolution is filtered, adopts moving phase: A 0.1% acetic acid/water, B 0.1% acetic acid/acetonitrile, analyze according to following program sample introduction under 214nm:
Weigh the target sample after freeze-drying, quality is 68.8mg, and crude yield is 91.8% as calculated, is improved largely than the yield of prior art only 60-70%.
4. the preparation of cetrorelix acetate IV
Cetrorelix crude product III is dissolved and filters, adopt rHPLC legal system for purifying, preparation condition is: 5um C18 preparative column, moving phase is A (0.1% acetic acid/water)/B (0.1% acetic acid/acetonitrile), flow velocity 9ml/min, determined wavelength 214nm, collects target sample and freeze-drying obtains cetrorelix polypeptide; By cetrorelix polypeptide 1% acetate dissolution, join stirring at room temperature absorption 1h in strong anion exchange resin, taking-up adsorption liquid is added acetic acid content and is sent freeze-drying to 5%, obtains cetrorelix acetate sterling 31.2mg through weighing, purity reaches 99.613%, and sterling yield is 41.2%; Take each 1mg of cetrorelix polypeptide turned before and after salt respectively, measure acetic acid content by official method, turning acetic acid content before salt is 1.56%, and after turning salt, acetic acid content is 6.9%, meet pharmacopoeial requirements (acetic acid content scope 4% ~ 8%), target compound cetrorelix acetate product is qualified.
Embodiment 2
Invention has been the contrast experiment of cetrorelix preparation method yield, namely 1. condensation reaction washs after normal condensation reaction 20 ~ 30min 2. after normal condensation reaction 20 ~ 25min, adds 0.5ml DIEA, washs after microwave aminoresin 25s; The yield synthesizing the cetrorelix crude product obtained through above-mentioned experimental technique is 1. 82.7%, 2. 91.8%, purified, turn salt and prepare cetrorelix acetate sterling 1. 24.1mg, 2. 31.5mg, and sterling yield is 1. 31.8%, 2. 41.2%.
Embodiment 3
Invention has been secondary and repeat synthesis, to determine the stability of experimental result, 1. condensation reaction washs after normal condensation reaction 20 ~ 30min 2. after normal condensation reaction 20 ~ 25min, adds 0.5ml DIEA, washs after microwave aminoresin 25s; The purity obtaining cetrorelix crude product reaches 83%, and crude yield is 1. 83.1%, 2. 92.4%, purified, turn salt and prepare cetrorelix acetate sterling 1. 24.3mg, 2. 31.3mg, and sterling yield is 1. 32.4%, 2. 40.7%; The exactness of proved invention experimental data and stability.
Test through above-described embodiment 2 and example 3, to draw after normal synthesis microwave reaction again, the yield of the cetrorelix acetate sterling prepared is higher, and effect is better.
Claims (8)
1. specificity Microwave synthesize prepares the method for cetrorelix acetate, it is characterized in that comprising the following steps: adopt solid-phase synthesis, initial vector selects Rink Amide-AM Resin, with HBTU/DIEA be condensation reagent and with Fmoc-protected amino acid for condensation raw material, amino acid corresponding in condensation cetrorelix aminoacid sequence successively, activated dose and the condensation reaction of condensing agent and the deprotection reaction of 20%Pip/DMF deprotection agent, obtain cetrorelix intermediate peptide I-aminoresin, recycle deprotection agent afterwards and take off D-Nal protecting group, carry out N and hold the reaction of acetylize end socket, cetrorelix intermediate peptide II-aminoresin is obtained with this, then through cutting precipitation reaction, finally obtain the thick peptide III of cetrorelix, product is through microwave, purifying, acetic acid turns salt technology and freeze-drying obtains cetrorelix acetate IV.
2. the method preparing cetrorelix acetate according to claim 1, it is characterized in that described initial aminoresin is Rink Amide-AM Resin, substitution value is 0.97mmol/g.
3. the method preparing cetrorelix acetate according to claim 1, it is characterized in that being activator and condensing agent with HBTU/DIEA in condensation reaction, condensation reaction time is 20min; Described deprotection agent is 20%Pip/DMF solution, and elution time is 15min.
4. the method preparing cetrorelix acetate according to claim 1; it is characterized in that the Fmoc-protected amino acid of solid-phase synthesis condensation is successively: Fmoc-Pro-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Leu-OH, Fmoc-D-Cit-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-D-Ala (3-pyridyl)-OH, Fmoc-D-Phe (4-Cl)-OH, Fmoc-D-2-Nal-OH; in described condensation reaction, amino acid charging capacity is thrown resin mole number by 1.5 times, and setting-up point is 25 DEG C.
5. the method preparing cetrorelix acetate according to claim 1, is characterized in that the acetylation reagent of acetylization reaction be volume ratio is aceticanhydride: the mixed solution of DIEA: DMF=1: 1: 4, the acetylization reaction time is 20min.
6. the method preparing cetrorelix acetate according to claim 1, it is characterized in that the cutting reagent 100ml formula cutting precipitation reaction is: 88ml TFA+5ml thioanisole+5ml water+2ml EDT, cleavage reaction temperature is 20 DEG C, and carries out the sedimentation of polypeptide crude product with the anhydrous diethyl ether of-20 DEG C of low temperature precoolings.
7. the method preparing cetrorelix acetate according to claim 1, it is characterized in that selecting 5 μm in the purifying process of polypeptide, C18,250mmI.D. × 20 [cm] preparative column carries out RPLC (rHPLC) method purifying, prepares Gradient program according to what optimize as follows
Purification of crude peptide III; Collect highly purified target sample and freeze-drying, join in chlorine type strong anion exchange resin with acetate dissolution polypeptide, to replace the trifluoroacetate in polypeptide products, obtain cetrorelix acetate IV sterling.
8. a species specificity Microwave synthesize prepares the method for cetrorelix acetate, it is characterized in that:
(1) method according to claims 1 to 7 obtains cetrorelix acetate sterling;
(2) specificity Microwave synthesize prepares the method for cetrorelix acetate, it is characterized in that: step of condensation in claim 4, after amino acid condensation reaction 20min, is used by aminoresin specific Microwave synthesize to react 25s.
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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CN106397542A (en) * | 2016-10-24 | 2017-02-15 | 合肥国肽生物科技有限公司 | Method for preparing triptorelin acetate through specific microwave synthesis |
CN106432427A (en) * | 2016-10-24 | 2017-02-22 | 合肥国肽生物科技有限公司 | Method for preparing gonadorelin acetate by virtue of specific microwave synthesis |
CN106478813A (en) * | 2016-10-24 | 2017-03-08 | 合肥国肽生物科技有限公司 | A kind of high-efficiency synthesis method of bivalirudin |
CN109467591A (en) * | 2018-12-27 | 2019-03-15 | 苏州天马医药集团天吉生物制药有限公司 | A kind of purification process of Cetrorelix |
CN110903352A (en) * | 2019-12-27 | 2020-03-24 | 中肽生化有限公司 | Preparation method of cetrorelix |
CN112933210A (en) * | 2019-12-11 | 2021-06-11 | 深圳翰宇药业股份有限公司 | Preparation method of cetrorelix freeze-dried pharmaceutical composition |
US11180533B2 (en) * | 2018-06-16 | 2021-11-23 | Biophore India Pharmaceuticals Private Limited | Process for the preparation of Cetrorelix acetate |
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CN104086632A (en) * | 2014-08-05 | 2014-10-08 | 杭州诺泰制药技术有限公司 | Method for preparing cetrorelix |
CN104277093A (en) * | 2013-07-12 | 2015-01-14 | 青岛贝泰克生物科技有限公司 | Method for preparing cetrorelix acetate by taking Rink Amide-AM Resin as carrier |
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CN101284863A (en) * | 2008-06-06 | 2008-10-15 | 吉尔生化(上海)有限公司 | Preparation method of solid phase synthesis cetrorelix |
CN104277093A (en) * | 2013-07-12 | 2015-01-14 | 青岛贝泰克生物科技有限公司 | Method for preparing cetrorelix acetate by taking Rink Amide-AM Resin as carrier |
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106397542A (en) * | 2016-10-24 | 2017-02-15 | 合肥国肽生物科技有限公司 | Method for preparing triptorelin acetate through specific microwave synthesis |
CN106432427A (en) * | 2016-10-24 | 2017-02-22 | 合肥国肽生物科技有限公司 | Method for preparing gonadorelin acetate by virtue of specific microwave synthesis |
CN106478813A (en) * | 2016-10-24 | 2017-03-08 | 合肥国肽生物科技有限公司 | A kind of high-efficiency synthesis method of bivalirudin |
US11180533B2 (en) * | 2018-06-16 | 2021-11-23 | Biophore India Pharmaceuticals Private Limited | Process for the preparation of Cetrorelix acetate |
CN109467591A (en) * | 2018-12-27 | 2019-03-15 | 苏州天马医药集团天吉生物制药有限公司 | A kind of purification process of Cetrorelix |
CN109467591B (en) * | 2018-12-27 | 2020-11-03 | 苏州天马医药集团天吉生物制药有限公司 | Purification method of cetrorelix |
CN112933210A (en) * | 2019-12-11 | 2021-06-11 | 深圳翰宇药业股份有限公司 | Preparation method of cetrorelix freeze-dried pharmaceutical composition |
CN110903352A (en) * | 2019-12-27 | 2020-03-24 | 中肽生化有限公司 | Preparation method of cetrorelix |
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