CN102731627A - Solid-phase synthesis method of Cilengitide - Google Patents

Solid-phase synthesis method of Cilengitide Download PDF

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Publication number
CN102731627A
CN102731627A CN2012101964109A CN201210196410A CN102731627A CN 102731627 A CN102731627 A CN 102731627A CN 2012101964109 A CN2012101964109 A CN 2012101964109A CN 201210196410 A CN201210196410 A CN 201210196410A CN 102731627 A CN102731627 A CN 102731627A
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Prior art keywords
fmoc
emd121974
wang resin
otbu
phase synthesis
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CN102731627B (en
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杨毅跃
姚程成
康国伟
鲁尧
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Yancheng Kaili Pharmaceutical Co., Ltd.
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WUXI KAILI PHARMACEUTICAL CO Ltd
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    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00Technologies relating to chemical industry
    • Y02P20/50Improvements relating to the production of bulk chemicals
    • Y02P20/55Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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

A kind of solid phase synthesis process of EMD121974
Technical field
The present invention relates to a kind of method of solid phase cyclization technology synthetic cyclic peptide, especially design the synthetic route of EMD121974.
Background technology
EMD121974 (Cilengitide), the Chinese another name: ring (L-arginyl glycyl-L-aspartoyl-D-phenylalanyl-N-methyl-L-valyl) is an a kind of new classification cancer therapy drug of synthetic.Merkel company discovers that EMD121974 amalgamation radiotherapy (merging to reach assists TM to add radiotherapy) possibly prolong lifetime; Simultaneously integrate plain supressor antitumor drug as first; Got into the III clinical trial phase, its important mechanism is to grow targeting that the blood supply structure of nutrition, the growth of promotion cancer cell is provided in tumour and for tumour through line artery.
The EMD121974 molecular formula is: C 27H 40N 8O 7, have following structure:
The preparation method of cyclic peptide mainly contains liquid phase synthesis process, solid phase synthesis precursor peptide cyclization process, process for solid phase synthesis in liquid phase at present; Wherein preceding two kinds of synthesis techniques all are the cyclisation in liquid phase of synthetic precursor peptide, and this method needs reactant in extremely rare solvent, to react (10 -3~10 -4Mol/L), and intermolecular be prone to react generation line style or cyclic polymer, greatly reduced the cyclisation yield, bring trouble for follow-up purifying, and in large-scale production, produce a large amount of waste liquids, be unfavorable for suitability for industrialized production.In conjunction with the structure of EMD121974, utilize the false rare principle of benefit of solid phase, developed a kind of efficient cyclization reaction, the cyclisation time shortens to 20%~30% of liquid phase cyclisation, and the 2%-8% of solvent as liquid phase used in reaction.
Summary of the invention
The synthesis technique of the solid phase cyclization technology that the present invention relates to that a kind of high yield, low cost, reaction conditions are gentle, environmental pollution is little, can large-scale industrial production.
This programme mainly may further comprise the steps:
1. by substitution degree Wang Resin and Fmoc-L-Asp-OtBu prepared in reaction Fmoc-L-Asp (the OtBu)-Wang Resin of 0.4~0.9mmol/g.
2. through solid phase synthesis method 4 amino acid that contain Fmoc of coupling residue successively progressively, obtain EMD121974 precursor peptide A-Wang Resin.
3. in EMD121974 precursor peptide A-Wang Resin, add the reagent that takes off Fmoc and OtBu respectively, obtain EMD121974 precursor peptide B-Wang Resin.
4. in DMF or DCM, add EMD121974 precursor peptide B-Wang Resin, cyclization reagent, through reaction EMD121974-Wang Resin.
5. cracking, purifying, lyophilize obtains pure EMD121974.
The solid phase cyclization synthesis technique of above-mentioned EMD121974, wherein
The described Fmoc-L-Asp of step 1) (OtBu)-Wang Resin, be Fmoc-L-Asp-OtBu and Wang Resin in DCM or DMF, under the DIC+HOBT+DMAP effect, generate.
Step 2) use solvent to be DMF or DCM in, coupling reagent is C+D+E, and wherein C is HOAt or HOBt, and D is HBTU or HATU or TBTU, and E is DIEA; The amino acid of described Fmoc protection is respectively: Fmoc-Gly-OH, Fmoc-L-Arg (Mtr)-OH, Fmoc-N-Me-L-Val, Fmoc-D-Phe-OH; The deprotection agent of Fmoc protection base is the piperidines-DMF solution of volume ratio 25%.
The deprotection base reagent that step 3) adds is respectively: it is 5~8h that the piperidines of volume ratio 25%-DMF solution and PhOH-DCM solution take off the OtBu reaction times.
The step 4) cyclization reagent is DPPA+DIEA or DPPA+NMM or DIC+HOBT or C+D+E, and wherein C is HOAr or HOBt, and D is HBTU or HATU or TBTU, and E is DIEA.
The step 5) lytic reagent is: TFA/H 2O/TlS.
Compare with existing traditional liquid phase cyclization reaction, that technology of the present invention has is simple to operate, yield is high, aftertreatment is simple, the reaction times is short, produce few, the low cost and other advantages of waste liquid, has a wide range of applications in cyclic peptide medicinal design synthesis technique field.
Description of drawings
Fig. 1 is a process flow sheet of the present invention.
Embodiment
Instance given below is necessary to point out at this that the present invention does not receive present method as unique restriction so that the present invention is specifically described, in this area the present invention is made some non-intrinsically safes and change and adjust, and all belongs to protection scope of the present invention.
The meaning of the abbreviation representative that in these claims and specification sheets, occurs is a following table:
Fmoc 9-fluorenylmethyloxycarbonyl
HOBt I-hydroxybenzotriazole
HOAt N-hydroxyl-7-azo benzotriazole
HATU 2-(7-azo benzotriazole)-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester
NMM N-methylmorpholine
Mtr 2,3,6-trimethylammonium-4-methoxy benzenesulfonyl
OtBu Tert.-butoxy
DMF N, dinethylformamide
DCM Methylene dichloride
DIC N, N '-DIC
HBTU 2-(7-azo benzotriazole)-tetramethyl-urea phosphofluoric acid ester
TBTU O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid
TFA Trifluoroacetic acid
DPPA Diphenyl phosphoryl azide
DMAP Dimethylamino pyridine
DIEA N, the N-diisopropylethylamine
TIS Tri isopropyl silane
PhOH Phenol
The specific embodiment explanation
Embodiment 1
The preparation of Fmoc-L-Asp (OtBu)-Wang Resin
The Wang Resin that takes by weighing the 10g substitution degree and be 0.5mmol/g joins in the reactor drum, adds an amount of DCM, and swelling 30min takes out DCM; 6.17g Fmoc-L-Asp-OtBu, DIC 2.40ml, HOBT2.1g are dissolved among the 30ml DMF; At 0-5 ℃ of activation 15min, activation solution is joined in the reactor drum that contains Wang Resin, behind the reaction 10min; Add DMAP 0.18g again, at 0~30 ℃ of reaction 1~5h.After reaction finishes, add sealing Wang Resin unreacted hydroxylation reagent diacetyl oxide 1ml and pyridine 0.5ml, behind the capping 1h, DMF, DCM, the CH of 80ml used in washing successively 3OH, DMF washing 2,1,1,2 times, each 1min.Through detecting, obtain the Fmoc-L-Asp that substitution degree is 0.47mmol/g (OtBu)-Wang Resin.
Embodiment 2
The EMD121974 precursor:
The preparation of A-Wang Resin (Fmoc-D-Phe-N-Me-L-Val-L-Arg (Mtr)-Gly-L-Asp (OtBu)-Wang Resin)
Fmoc-L-Asp (OtBu)-Wang Resin is joined in the reactor drum, behind DMF swelling 30min, take out solvent, the piperidines-DMF that adds 80ml 25% reacts 5min, and 80ml DMF washs 1 time (3min), and the piperidines-DMF that adds 80ml 25% reacts 15min; DMF, DCM, the CH of 80ml used in washing successively 3OH, DMF washing 2,1,1,2 times, each 1min; With 4.45g Fmoc-Gly-OH, 5.68g HBTU, 2.03g HOBt, be dissolved among the DMF of 30ml, dissolve the back and added DIEA 2.45ml; 0~5 ℃ of activation 15min; Activation solution is joined in the above-mentioned reactor drum, and behind reaction 1-3h under 0~30 ℃, reaction end detects with ninhydrin method.Adopt aforesaid method coupling Fmoc-L-Arg (Mtr)-OH, Fmoc-N-Me-L-Val, Fmoc-D-Phe-OH successively, finally obtain Fmoc-D-Phe-N-Me-L-Val-L-Arg (Mtr)-Gly-L-Asp (OtBu)-Wang Resin.
Embodiment 3
EMD121974 precursor peptide: the preparation of B-Wang Resin (D-Phe-N-Me-L-Val-L-Arg (Mtr)-Gly-L-Asp-Wang Resin)
With volume ratio is that piperidines-DMF of 25% is the Fmoc deprotection agent of Fmoc-D-Phe-N-Me-L-Val-L-Arg (Mtr)-Gly-L-Asp (OtBu)-Wang Resin; Add piperidines-DMF 80ml of 25% first time; Reaction 5min, 80ml DMF washs 1 time (3min), adds piperidines-DMF 80ml of 25% for the second time; Behind the reaction 15min, DMF, DCM, the CH of 80ml used in washing successively 3OH, DMF washing 2,1,1,2 times, each 1min gets D-Phe-N-Me-L-Val-L-Arg (Mtr)-Gly-L-Asp (OtBu)-Wang Resin after washing finishes.
80% the PhOH-DCM solution that adds volume ratio and be 100ml takes off OtBu with the TFA of catalytic amount, reacts 8h; DMF, DCM, the CH of 80ml used in washing successively 3OH, DMF washing 2,1,1,2 times, each 1min gets D-Phe-N-Me-L-Val-L-Arg (Mtr)-Gly-L-Asp-Wang Resin.
Embodiment 4
The preparation of EMD121974-Wang Resin (Cyclo (D-Phe-N-Me-L-Val-L-Arg-Gly-L-Asp)-Wang Rsin)
In above-mentioned reactor drum, add cyclization reagent 3.9g DPPA, 2.5ml DIEA (reactant cyclization reagent amount of substance ratio is 1: 3), at 10~40 ℃ of reaction 3h, the multiple cyclization reagent reaction 3~5h (reaction end detects with ninhydrin method) that throws once above-mentioned equivalent; DMF, DCM, the CH of 80ml used in washing successively 3OH washing 2,1,3 times, each 3min gets Cyclo (D-Phe-N-Me-L-Val-L-Arg-Gly-L-Asp)-Wang Rsin.
Embodiment 5
The preparation of EMD121974 (Cyclo (D-Phe-N-Me-L-Val-L-Arg-Gly-L-Asp))
In above-mentioned reactor drum, add the TFA/H of lytic reagent 120ml again 2Behind O/TlS (volume ratio is 95: 2.5: 2.5) the reaction 3h, suction filtration is removed resin, and filtrating slowly joins in the no water-ice ether; Static 2-5h, high speed centrifugation obtain thick peptide, prepare through high-pressure liquid phase; Lyophilize gets smart EMD121974; Its purity>99.5%, single impurity<0.2%, total recovery reaches 63%.
Choosing substitution degree in the present embodiment is the Wang Resin of 0.5mmol/g, and can also choose substitution degree is the arbitrary Wang Resin and Fmoc-L-Asp-OtBu prepared in reaction Fmoc-L-Asp (the OtBu)-Wang Resin of 0.4~0.9mmol/g scope.All can realize technical scheme of the present invention, and obtain technique effect of the present invention.
Above content is an EMD121974 and become one of best preferred version of route; And to further explain that the present invention did; But can not assert that practical implementation of the present invention is only limited to these explanations; Under the prerequisite that does not break away from the present invention's design, can also make some simple deductions and replacement, all should be regarded as protection domain of the present invention.

Claims (6)

1. the method for the solid phase synthesis of an EMD121974 may further comprise the steps:
1) by substitution degree is Wang Resin and Fmoc-L-Asp-OtBu prepared in reaction Fmoc-L-Asp (the OtBu)-Wang Resin of 0.4~0.9mmol/g;
2) through 4 Fmoc amino acid of the residue of solid-phase synthesis coupling successively, obtain EMD121974 precursor peptide A-Wang Resin;
3) in EMD121974 precursor peptide A-Wang Resin, add the reagent that takes off Fmoc and OtBu respectively, obtain EMD121974 precursor peptide B-Wang Resin;
4) add cyclization reagent, react EMD121974-Wang Resin;
5) cracking, purifying, lyophilize obtains pure EMD121974.
2. the method for the solid phase synthesis of EMD121974 according to claim 1 is characterized in that, Fmoc-L-Asp-OtBu and Wang Resin be prepared in reaction Fmoc-L-Asp (OtBu)-Wang Resin under the DIC+HOBT+DMAP effect.
3. the method for the solid phase synthesis of EMD121974 according to claim 1; It is characterized in that; Solid phase synthesis EMD121974 precursor peptide A-Wang Resin, wherein Fmoc-amino acid is respectively Fmoc-Gly-OH, Fmoc-L-Arg (Mtr)-OH, Fmoc-N-Me-L-Val-OH and Fmoc-D-Phe-OH.
4. according to the method for the solid phase synthesis of any described EMD121974 of claim of claim 1-3, it is characterized in that the solvent of use is dry DMF, DCM, CH 3OH, coupling agent are C+D+E, and wherein C is HOAt or HOBt, and D is HBTU or HATU or TBTU, and E is DIEA, and Fmoc protection base is piperidines-DMF (DCM) solution of 20%~25% for volume ratio on the deaminizating, and the reagent that takes off OtBu is the PhOH solution that contains catalytic amount TFA.
5. the method for the solid phase synthesis of EMD121974 according to claim 1; It is characterized in that, synthetic EMD121974-Wang Resin, wherein cyclization reagent is DPPA+DIEA or DPPA+NMM or DIC+HOBT or C+D+E; Wherein C is HOAt or HOBt; D is HBTU or HATU or TBTU, and E is DIEA, and taking off Side chain protective group and lytic reagent is TFA/H 2O/TIS.
6. according to the method for the solid phase synthesis of any described EMD121974 of claim of claim 1-5, it is characterized in that the activation of amino acids temperature is 0~5 ℃, coupling, deprotection base, cracking and cyclization temperature are 0~30 ℃.
CN201210196410.9A 2012-06-14 2012-06-14 A kind of solid phase synthesis process of cilengitide Active CN102731627B (en)

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Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
NOTHELFER 等: "Identification and characterization of a peptide with affinity to head and neck cancer", 《THE JOURNAL OF NUCLEAR MEDICINE》 *
SABATINO G等: "Assessment of 6CL-HOBT-based coupling reagents in solid-phase cyclopeptide synthesis", 《PEPTIDE REVOLUTION:GENOMICS,PROTEMICS & THERAPEUTICS》 *
THUMSHIRN 等: "Multimeric cyclic RGD peptides as potential tools for tumor targeting:solid-phase peptide synthesis and chemoselective oxime ligation", 《CHEM.EUR.》 *
徐岳义 等: "有机合成中肽缩合试剂的发展概况(二)", 《宁波化工》 *
白东鲁 等: "《高等药物化学》", 30 November 2011 *
郑彦慧 等: "Fmoc保护氨基酸与Wang树脂的缩合反应", 《高等学校化学学报》 *

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