CN104017058A - Method for preparing ganirelix acetate - Google Patents
Method for preparing ganirelix acetate Download PDFInfo
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- CN104017058A CN104017058A CN201410265365.7A CN201410265365A CN104017058A CN 104017058 A CN104017058 A CN 104017058A CN 201410265365 A CN201410265365 A CN 201410265365A CN 104017058 A CN104017058 A CN 104017058A
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- 238000000034 method Methods 0.000 title claims abstract description 27
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- AHZJKOKFZJYCLG-UHFFFAOYSA-K trifluoromethanesulfonate;ytterbium(3+) Chemical compound [Yb+3].[O-]S(=O)(=O)C(F)(F)F.[O-]S(=O)(=O)C(F)(F)F.[O-]S(=O)(=O)C(F)(F)F AHZJKOKFZJYCLG-UHFFFAOYSA-K 0.000 claims abstract description 12
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Classifications
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- 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
Abstract
The invention relates to a method for preparing ganirelix acetate. The method comprises the following steps: A) by using an amino resin as an initial resin, sequentially coupling amino acids with side chain protection according to the ganirelix acetate peptide sequence by an Fmoc solid-phase synthesis process, wherein the 6th and 8th amino acids for coupling in the peptide sequence respectively adopt Fmoc-D-Lys(Dde)-OH and Fmoc-Lys(Dde)-OH; B) after finishing coupling, removing the Fmoc protecting group, and carrying out acetylation reaction at the N terminal; C) removing the Dde protecting group by using a mixed solution composed of hydrazine hydrate and DMF (N,N-dimethylformamide) in a volume ratio of 3:97; D) adding N,N'-diethylcarbodiimide and ytterbium trifluoromethanesulfonate into the resin to carry out side chain modification reaction to form an Et-protected guanido group; and E) cracking, purifying and freeze-drying to obtain the ganirelix acetate. The technique has the advantages of simple synthesis process, low cost and higher yield. The technique directly synthesizes and modifies effective components on the carrier, thereby avoiding directly buying or synthesizing expensive non-natural amino acids by liquid phase, effectively enhancing the yield and lowering the production cost.
Description
Technical field
The present invention relates to a kind of preparation method of polypeptide drug, is a kind of synthetic decapeptide GnRH(endogenous gonadoliberin) preparation method of simulating peptide ganirelix acetate.
Background technology
Ganirelix acetate, illustrious name is: Ganirelix, structural formula is as follows:
Peptide sequence is:
The aminoacid sequence of figure-1 Ganirelix
Ganirelix (Ganirelix, AntagonTM) be a synthetic decapeptide GnRH(endogenous gonadoliberin of being developed by Merck) simulating peptide, the 1st, 2,3,6,8 of natural GnRH and 10 amino acids are replaced, natural GnRH is had to higher antagonistic action.
Research shows, the GnRH acceptor of this product on can competitive binding pituitary body gonadotroph, the release of rapid, reversible inhibition gonad-stimulating hormone.To pituitary body LH(luteotropic hormone) secretion restraining effect be better than FSH(follicular stimulating hormone) control.After drug withdrawal, the secretion of LH and FSH can recover normal in 48 hours.On July 29th, 1999 by U.S. FDA approval for accepting the women of controlled super ovulation supplementary reproduction treatment, control LH(luteotropic hormone) peak is too early.
US4801577, US5212288 and US5767082 disclose a kind of solid-phase synthesis of ganirelix acetate of Boc strategy, however this technique is backward, and waste liquid is many, and cost is expensive, and yield is low, and environmental pollution is large, is unfavorable for scale operation.
CN102584945A discloses the preparation method of a kind of ganirelix acetate of a kind of Fomc strategy, by Fmoc solid-phase synthesis, prepares ganirelix acetate, but due to Fomc-homoArg (Et)
2-OH and Fomc-D-homoArg (Et)
2-OH preparation needs preparation separately, synthesis and purification more complicated, and isomer is not easy to control, and synthesis cycle is long, and cost is high, and yield is low, and impurity is many, is not suitable for suitability for industrialized production.
For this reason, the synthetic method of inventor's Dichlorodiphenyl Acetate Ganirelix is studied, thereby has obtained technical scheme of the present invention.
Summary of the invention
The solid phase synthesis process that the object of this invention is to provide a kind of ganirelix acetate.The technical issues that need to address of the present invention are: Fomc-homoArg (Et)
2-OH and Fomc-D-homoArg (Et)
2-OH preparation needs preparation separately, synthesis and purification more complicated, and isomer is not easy to control, and synthesis cycle is long, and cost is high, and yield is low, and impurity is many, is not suitable for suitability for industrialized production.
Synthetic route of the present invention is as shown in Figure 1: take aminoresin as initial resin, by Fmoc solid-phase synthesis, the amino acid according to ganirelix acetate peptide order successively coupling with side chain protected, wherein peptide order 6, 8 amino acids couplings adopt respectively Fmoc-D-Lys (Dde)-OH and Fmoc-Lys (Dde)-OH, after coupling completes, remove Fmoc protecting group, then N end carries out acetylization reaction, the mixing solutions that the hydrazine hydrate that employing volume ratio is 3:97 and DMF form removes Dde protecting group, then in resin, add N, N'-diethyl carbodiimide and Ytterbiumtriflate carry out side chain modification reaction, form the guanidine radicals of Et protection, last cracking, purifying, freeze-drying obtains ganirelix acetate.
In the present invention, some conventional abbreviations have following implication;
Fmoc: fluorenylmethyloxycarbonyl
Fmoc-AA: the amino acid of fluorenylmethyloxycarbonyl protection
DIC: N, N '-di-isopropyl carbodiimide
DCC: N, N '-dicyclohexylcarbodiimide
PyBOP: phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus
HATU: 2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester
HOBt: 1-hydroxybenzene a pair of horses going side by side triazole
HOSu: N-hydroxy-succinamide
TBu: the tertiary butyl
Trt: trityl
Boc: tertbutyloxycarbonyl
Pbf: 2,2,4,6,7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl
Dde: N-1-(4,4-dimethyl-2,6-dioxy cyclohexylene-1) ethyl
HomoArg: homoarginine
Tyr: tyrosine
Pro: proline(Pro)
Leu: leucine
Lys: Methionin
Ser: Serine
D-2-Nal-OH:D-2-naphthylalanine
4-Chloro-D-Phe-OH:D-4-chlorophenylalanine
3-(3-pyridyl)-D-alanine:D-3-(3-pyridyl)-L-Ala
DMF: N, N '-dimethyl formamide
Ac
2o: acetic anhydride
Py: pyridine
MeOH: methyl alcohol
DCM: methylene dichloride
NMP: N-Methyl pyrrolidone
DMSO: dimethyl sulfoxide (DMSO)
TFA: trifluoracetic acid
EDT: dithioglycol
Piperidine: hexahydropyridine
DMAP:4-Dimethylamino pyridine
DIEA: N, N '-diisopropylethylamine
TMP: 2，4，6-trimethylpyridine
OTf: trifluoromethanesulfonic acid
Yb: ytterbium
The invention provides a kind of synthetic method of ganirelix acetate, its step is as follows for this reason:
Step 1, take aminoresin as initial resin, by Fmoc solid-phase synthesis, according to ganirelix acetate peptide order successively coupling, there is the amino acid of side chain protected, wherein 6, the 8 amino acids couplings of peptide order adopt respectively Fmoc-D-Lys (Dde)-OH and Fmoc-Lys (Dde)-OH;
Step 2, after coupling completes, removes Fmoc protecting group, and then N end carries out acetylization reaction;
Step 3, the mixing solutions that the hydrazine hydrate that employing volume ratio is 3:97 and DMF form removes Dde protecting group;
Step 4 adds N in resin, and N'-diethyl carbodiimide and Ytterbiumtriflate carry out side chain modification reaction, forms the guanidine radicals of Et protection;
Step 5, cracking, purifying, freeze-drying, obtains ganirelix acetate.
Wherein, the solid phase synthesis process described in step 1,1) adopt the Fmoc-aminoresin of 0.50 ~ 1.0 mmol/g; 2) adopt going that the piperidines be 1:4 by volume ratio and DMF form to protect liquid to remove the Fmoc protecting group on aminoresin; 3) under the existence of coupling agent system, NH
2-resin and Fmoc-D-Ala-OH coupling obtain Fmoc-D-Ala-NH-resin; 4) repeating step 2), 3), according to ganirelix acetate main chain peptide order successively coupling, have the amino acid of N end Fmoc protection and side chain protected, wherein 6,8 amino acids couplings adopt respectively Fmoc-D-Lys(Dde)-OH and Fmoc-Lys(Dde)-OH; 5) after coupling completes, remove the Fmoc protecting group of peptide chain N end, adopt the mixing solutions of aceticanhydride, alkali and DMF to carry out acetylization reaction; 6) mixing solutions that adopts hydrazine hydrate that volume ratio is 3:97 and DMF to form removes Fmoc-D-Lys(Dde)-OH and Fmoc-Lys(Dde) Dde protecting group on-OH; 7) adopt N, N'-diethyl carbodiimide and Ytterbiumtriflate carry out side chain modification reaction, form the guanidine radicals of Et protection; 8) cracking, purifying, freeze-drying, obtains ganirelix acetate.
Wherein, the acetylization reaction described in step 2 is: the mixing solutions reaction 1 ~ 2h that adopts aceticanhydride, N-methylmorpholine and DMF combination that volume ratio is 1:1:8.。
Wherein, wherein, resin, N described in step 4, N'-diethyl carbodiimide and Ytterbiumtriflate mol ratio are 1:2:0.2% ~ 1:2:5%, temperature of reaction is 25 ~ 35 ℃; 2 ~ 3 hours reaction times, more preferably, Ac-D-Nal-4-Chloro-D-Phe-3-(3-Pyridyl)-D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys-Leu-Lys-Pro-D-Ala-NH-resin, N, N'-diethyl carbodiimide and Ytterbiumtriflate mol ratio are 1:2:1%, temperature of reaction: 25 ℃, the reaction times is 3 hours.
Method of the present invention obtains through screening, and screening process is as follows:
1, material (decapeptide resin is Ac-D-Nal-4-Chloro-D-Phe-3-(3-Pyridyl)-D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys-Leu-Lys-Pro-D-Ala-NH-resin, N, N'-diethyl carbodiimide and Ytterbiumtriflate) mol ratio:
Decapeptide resin, N, N'-diethyl carbodiimide and Ytterbiumtriflate mol ratio are 1:2:0.5%; 1:2:1 %.
2, the selection of temperature of reaction:
25
oc and 35
oc
3, the selection in reaction times:
2 hours and 3 hours.
8 kinds of experiment conditions have been proposed for this reason:
Experiment condition 1: get 2.98g (1.0mmol) Ac-D-Nal-4-Chloro-D-Phe-3-(3-Pyridyl)-D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys-Leu-Lys-Pro-D-Ala-NH-resin; 197.2mg (2.0 mmol) N, N'-diethyl carbodiimide; (3.3mg 0.005 mmol) Yb (OTf)
3add 10mlTHF under anhydrous and oxygen-free condition 25
oc reaction 2 hours, with DMF washing 6 times, DCM washing 3 times, MeOH washing 3 times, DCM washing 3 times, MeOH washing 3 times, drains and obtains Ganirelix peptide resin, cracking, purifying, freeze-drying, obtains ganirelix acetate essence peptide.
Experiment condition 2-8, experimental implementation as shown in experiment condition 1, the experimental result of different experiment conditions as shown in Table 1 below:
The experimental result of the experiment condition that table 1 is different
| Experiment condition | Molar ratio of material | Temperature | Time | Total recovery | Purity |
| Experiment condition 1 | 1:2:0.5% | 25℃ | 2 hours | 30% | 99.35% |
| Experiment condition 2 | 1:2:1% | 25℃ | 2 hours | 35% | 99.38% |
| Experiment condition 3 | 1:2:0.5% | 35℃ | 2 hours | 49% | 99.48% |
| Experiment condition 4 | 1:2:1% | 35℃ | 2 hours | 51% | 99.50% |
| Experiment condition 5 | 1:2:0.5% | 25℃ | 3 hours | 38% | 99.41% |
| Experiment condition 6 | 1:2:1% | 25℃ | 3 hours | 55% | 99.65 % |
| Experiment condition 7 | 1:2:0.5% | 35℃ | 3 hours | 52% | 99.54% |
| Experiment condition 8 | 1:2:1% | 35℃ | 3 hours | 40% | 99.50 % |
Above result shows, yield and the purity of experiment condition 6 are all the highest, so experiment condition 6 is optimal conditionss.
Compared to the prior art method of the present invention has obvious advantage, and relevant contrast experiment is as shown in table 2 below:
Table 2 contrast and experiment
| Patent | Total recovery/% | Purity/% |
| The technology of the present invention | 55% | 99.65% |
| CN 102584945 A | 40% | 99.35% |
The invention has the beneficial effects as follows: the direct effective member of synthetic modification on carrier, avoided direct purchase or liquid phase to synthesize expensive alpha-non-natural amino acid, effectively improved yield, reduced production costs.
Accompanying drawing explanation
Fig. 1 synthetic route of the present invention;
The HPLC spectrogram of the thick peptide of Fig. 2 ganirelix acetate;
The HPLC spectrogram of Fig. 3 ganirelix acetate essence peptide;
Fig. 4 ganirelix acetate essence peptide mass spectrogram.
Embodiment
Further illustrate by the following examples the present invention.
Particularly, be respectively purchased amino acid and amino acid fragment, and be respectively purchased resin about what relate in embodiment below, its manufacturer and marque are as follows:
Fmoc protecting group amino acid starting material and AM resin are conventional commercial reagent (producer: the biochemical (Shanghai) Co., Ltd. of gill; Chemical pure).
Organic solvent and other raw material sources are commercially available product (producer: Chemical Reagent Co., Ltd., Sinopharm Group and A Faaisha (Tianjin) Chemical Co., Ltd.; Chemical pure).
In addition, " concentrated by rotary evaporation " mentioned in embodiment below and " freeze-drying " and mensuration HPLC and mass spectrographic condition and equipment used model and manufacturer are described as follows:
Concentrated by rotary evaporation equipment: Rotary Evaporators R-200/205(Switzerland Buchi (cloth is strange) company);
Concentrated by rotary evaporation condition: at 30 ℃, concentrated by rotary evaporation under vacuum (0.1Mpa) condition, concentrated after volume revolve steam before cumulative volume below 75%.
Freeze-drier: Freeze Drying Equipment FD-3(Beijing Bo Yikang laboratory apparatus company limited);
Lyophilisation condition: freeze-drying dish is put into freezer compartment of refrigerator (20 ℃), pre-freeze 6 hours.Open Freeze Drying Equipment, open refrigeration, precooling more than 30 minutes, arranges freeze-drying curve as follows:
First paragraph :-27 ℃ of operations 16 hours; Second segment :-5 ℃ of operations 4 hours; The 3rd section: 5 ℃ of operations 2 hours; The 4th section: 30 ℃ of operations 16 hours.
HPLC:Dionex high performance liquid chromatograph; With octadecylsilane chemically bonded silica (5 μ m, 250 * 4.6mm), it is weighting agent; Take 0.1%TFA solution as mobile phase A, take acetonitrile as Mobile phase B, carry out gradient elution; Flow velocity is per minute 1.0ml; Detection wavelength is 220nm; 30 ℃ of column temperatures.Get need testing solution 20 μ l, injection liquid chromatography, records color atlas.
Mass spectrum: MALDI-TOF-MS ground substance assistant laser desorption ionization flight time mass spectrum; Instrument model is AUTO FLEX SPEED TOF-TOF.
Embodiment mono-: Yb (OTf)
3synthetic
By purity, be that 39.41g 99.9% ytterbium oxide (100mmol) joins in the mixing 1000mL solution that trifluoromethanesulfonic acid that volume ratio is 1:1 and water forms, reflux 1h, then filter, remove not reacted ytterbium oxide, decompression dewaters and obtains the Ytterbiumtriflate with crystal water, finally, 200 ℃ of vacuum hydro-extractions 36 hours, obtain 58.90g Yb (OTf)
3.
Embodiment bis-: Ac-D-Nal-4-Chloro-D-Phe-3-(3-Pyridyl)-D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys (Dde)-Leu-Lys (Dde)-Pro-D-Ala-NH-Rink amide AM resin resin synthetic
Taking 1.38 g substitution values is the Rink amide AM resin (1 mmol) of 0.728 mmol/g, join in solid state reaction post, with DMF washing 1 time, with DCM swelling resin after 30 minutes, what adopt that piperidines that volume ratio is 1:4 and DMF form goes to protect liquid reaction 5 minutes, DMF washing 1 time, what adopt that piperidines that volume ratio is 1:4 and DMF form goes to protect liquid reaction 10 minutes, DMF washing 6 times, take 0.93 g Fmoc-D-Ala-OH(3 mmol), 0.40 g HOBt(3 mmol) adding volume ratio is DCM and the DMF mixing solutions of 1:1, under ice-water bath, add 0.46 ml DIC(3 mmol) activation after, add in the above-mentioned reaction column that resin is housed, under room temperature, react 2 hours, with ninhydrin method, detect judgement reaction end, if resin water white transparency, represent to react completely, resin colour developing, represents reaction not exclusively, need to react 1 hour again, and this judging criterion is applicable to ninhydrin method, detect judgement reaction end in follow-up amino acid coupling.Repeat the step that the above-mentioned Fmoc of removing protected and added corresponding amino acid coupling, according to peptide order, complete successively Fmoc-Pro-OH, Fmoc-Lys(Dde)-OH, Fmoc-Leu, Fmoc-D-Lys (Dde)-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-3-(3-pyridyl)-D-alanine, Fmoc-4-Chloro-D-Phe-OH, the coupling of Fmoc-D-Nal-OH, obtain Ac-D-Nal-4-Chloro-D-Phe-3-(3-Pyridyl)-D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys (Dde)-Leu-Lys (Dde)-Pro-D-Ala-NH-Rink amide AM resin resin.
Embodiment tri-: Ac-D-Nal-4-Chloro-D-Phe-3-(3-Pyridyl)-D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys (Dde)-Leu-Lys (Dde)-Pro-D-Ala-NH-Rink amide MBHA resin resin synthetic
Taking 1.37 g substitution values is the Rink amide MBHA resin (1 mmol) of 0.73 mmol/g, join in solid state reaction post, with DMF washing 1 time, with DCM swelling resin after 30 minutes, what adopt that piperidines that volume ratio is 1:4 and DMF form goes to protect liquid reaction 5 minutes, DMF washing 1 time, what adopt that piperidines that volume ratio is 1:4 and DMF form goes to protect liquid reaction 10 minutes, DMF washing 6 times, take 0.93 g Fmoc-D-Ala-OH(3 mmol), 0.40 g HOBt(3 mmol) adding volume ratio is DCM and the DMF mixing solutions of 1:1, under ice-water bath, add 0.46 ml DIC(3 mmol) activation after, add in the above-mentioned reaction column that resin is housed, under room temperature, react 2 hours, with ninhydrin method, detect judgement reaction end, if resin water white transparency, represent to react completely, resin colour developing, represents reaction not exclusively, need to react 1 hour again, and this judging criterion is applicable to ninhydrin method, detect judgement reaction end in follow-up amino acid coupling.Repeat the step that the above-mentioned Fmoc of removing protected and added corresponding amino acid coupling, according to peptide order, complete successively Fmoc-Pro-OH, Fmoc-Lys(Dde)-OH, Fmoc-Leu, Fmoc-D-Lys (Dde)-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-3-(3-pyridyl)-D-alanine, Fmoc-4-Chloro-D-Phe-OH, the coupling of Fmoc-D-Nal-OH, obtain Ac-D-Nal-4-Chloro-D-Phe-3-(3-Pyridyl)-D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys (Dde)-Leu-Lys (Dde)-Pro-D-Ala-NH-Rink amide MBHA resin resin.
Embodiment tetra-: Ac-D-Nal-4-Chloro-D-Phe-3-(3-Pyridyl)-D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys-Leu-Lys-Pro-D-Ala-NH-Rink amide AM resin resin synthetic
Measure hydrazine hydrate 3ml; add 97mlDMF; be made into the mixing solutions that hydrazine hydrate that volume ratio is 3:97 and DMF form; add reaction tubes; at room temperature react after 15min; with DMF washing once; adding volume ratio is that the hydrazine hydrate of 3:97 and the mixing solutions of DMF composition go to protect after 30min again; with DMF washing 6 times, obtain Ac-D-Nal-4-Chloro-D-Phe-3-(3-Pyridyl)-D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys-Leu-Lys-Pro-D-Ala-NH-Rink amide AM resin resin.
Embodiment five: Ac-D-Nal-4-Chloro-D-Phe-3-(3-Pyridyl)-D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys-Leu-Lys-Pro-D-Ala-NH-Rink amide MBHA resin resin synthetic
Measure hydrazine hydrate 3ml; add 97mlDMF; be made into the mixing solutions that hydrazine hydrate that volume ratio is 3:97 and DMF form; add reaction tubes; at room temperature react after 15min; with DMF washing once; adding volume ratio is that the hydrazine hydrate of 3:97 and the mixing solutions of DMF composition go to protect after 30min again; with DMF washing 6 times, obtain Ac-D-Nal-4-Chloro-D-Phe-3-(3-Pyridyl)-D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys-Leu-Lys-Pro-D-Ala-NH-Rink amide MBHA resin resin.
Embodiment six: Ganirelix peptide Rink amide AM resin resin synthetic
By the Ac-D-Nal-4-Chloro-D-Phe-3-obtaining in embodiment tetra-(3-Pyridyl)-D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys-Leu-Lys-Pro-D-Ala-NH-Rink amide AM resin (1.0mmol); 197.2mg N, N'-diethyl carbodiimide (2.0 mmol); 6.6 mg Yb (OTf)
3(0.01 mmol) adds 10mlTHF rousing under condition of nitrogen gas 25
oc reaction 3 hours, with DMF washing 6 times, DCM washing 3 times, MeOH washing 3 times, DCM washing 3 times, MeOH washing 3 times, drains and obtains Ganirelix peptide Rink amide AM resin resin 3.10g.
Embodiment seven: Ganirelix peptide Rink Amide MBHA resin resin synthetic
By the Ac-D-Nal-4-Chloro-D-Phe-3-obtaining in embodiment five (3-Pyridyl)-
D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys-Leu-Lys-Pro-D-Ala-NH-Rink Amide MBHA resin (1.0mmol); 197.2mg N, N'-diethyl carbodiimide (2.0 mmol); 6.6 mg Yb (OTf)
3(0.01 mmol) adds 10mlTHF rousing under condition of nitrogen gas 25
oc reaction 3 hours, with DMF washing 6 times, DCM washing 3 times, MeOH washing 3 times, DCM washing 3 times, MeOH washing 3 times, drains and obtains Ganirelix peptide Rink Amide MBHA resin resin 3.25g.
Embodiment eight: the thick peptide of Ganirelix synthetic
The 3.10 g Ganirelix peptide Rink amide AM resin resins of embodiment six are joined in three mouthfuls of round-bottomed flasks of 50 mL, be the TFA of 90:5:3:2 by volume, thioanisole, configuration lysate 31 mL of methyl-phenoxide and EDT combination, lysate is added in above-mentioned resin, room temperature reaction 2 hours, filter, with the resin after a small amount of TFA washing cracking 3 times, merging filtrate, concentrated, liquid after concentrated is joined in ice ether and precipitates 1 hour, centrifugal, anhydrous diethyl ether centrifuge washing 6 times, vacuum-drying, obtain thick peptide 1.36 g of Ganirelix, its HPLC spectrogram as shown in Figure 2, HPLC purity 92.68 %, synthesis yield 80.28 %.
Embodiment nine: the preparation of ganirelix acetate essence peptide
After thick peptide 1.36 g of Ganirelix in embodiment eight are dissolved with acetonitrile-water mixture, by C18 column purification, purification condition: moving phase is: A phase: 0.1 %TFA; B phase: acetonitrile; Gradient program is: 5 %B, in 60 minutes to 45%B; Detect wavelength 220 nm; Collect object peak cut.The condition of desalination: moving phase: A phase: the aqueous solution of 20 mmol/L ammonium acetates: acetonitrile=95:5; B phase: water: acetonitrile=95:5; C phase: 0.03 % vinegar aqueous acid: acetonitrile=95:5; D phase: 0.03 % vinegar aqueous acid: acetonitrile=50:50; Gradient program is: with mobile phase A Gradient elution 15 minutes, convert Mobile phase B Gradient elution to 10 minutes, convert moving phase C Gradient elution 10 minutes to, convert moving phase D Gradient elution 25 minutes to; Detect wavelength 220 nm; Collect object peak cut; Concentrated by rotary evaporation, freeze-drying obtains ganirelix acetate salt essence peptide 0.86g, its HPLC spectrogram as shown in Figure 3, HPLC purity 99.65%, purifying total recovery 63%, total recovery 55%.Its mass spectrum as shown in Figure 4, [M]
+: 1569.948, the accurate abundance maximum of theory of ganirelix acetate is: 1568.842, sample mass spectrum result conforms to theoretical molecular.
Claims (6)
1. the synthetic method of a ganirelix acetate, a kind of method of preparing ganirelix acetate is provided, provide and the invention provides a kind of preparation technology easy, cost is low, yield is higher that synthesizes, this technique is the effective member of synthetic modification on carrier directly, avoided direct purchase or liquid phase to synthesize expensive alpha-non-natural amino acid, effectively improved yield, reduced production costs, it is characterized in that, said method comprising the steps of:
Step 1, take aminoresin as initial resin, by Fmoc solid-phase synthesis, according to ganirelix acetate peptide order successively coupling, there is the amino acid of side chain protected, wherein 6, the 8 amino acids couplings of peptide order adopt respectively Fmoc-D-Lys (Dde)-OH and Fmoc-Lys (Dde)-OH;
Step 2, after coupling completes, removes Fmoc protecting group, and then N end carries out acetylization reaction;
Step 3, the mixing solutions that the hydrazine hydrate that employing volume ratio is 3:97 and DMF form removes Dde protecting group;
Step 4 adds N in resin, and N'-diethyl carbodiimide and Ytterbiumtriflate carry out side chain modification reaction, forms the guanidine radicals of Et protection;
Step 5, cracking, purifying, freeze-drying, obtains ganirelix acetate.
2. method according to claim 1, is characterized in that:
Wherein, the solid phase synthesis process described in step 1,1) adopt the Fmoc-aminoresin of 0.50 ~ 1.0 mmol/g; 2) adopt going that the piperidines be 1:4 by volume ratio and DMF form to protect liquid to remove the Fmoc protecting group on aminoresin; 3) under the existence of coupling agent system, NH
2-resin and Fmoc-D-Ala-OH coupling obtain Fmoc-D-Ala-NH-resin; 4) repeating step 2), 3), according to ganirelix acetate main chain peptide order successively coupling, have the amino acid of N end Fmoc protection and side chain protected, wherein 6,8 amino acids couplings adopt respectively Fmoc-D-Lys(Dde)-OH and Fmoc-Lys(Dde)-OH.
3. method according to claim 1, is characterized in that:
Wherein, the acetylization reaction described in step 2 is: the mixing solutions reaction 1 ~ 2h that adopts aceticanhydride, N-methylmorpholine and DMF combination that volume ratio is 1:1:8.
4. method according to claim 1, is characterized in that:
Wherein, resin, N described in step 4, N'-diethyl carbodiimide and Ytterbiumtriflate mol ratio are 1:2:0.2% ~ 1:2:5%, temperature of reaction is 25 ~ 35 ℃; 2 ~ 3 hours reaction times, more preferably, Ac-D-Nal-4-Chloro-D-Phe-3-(3-Pyridyl)-D-alanine-Ser (tBu)-Tyr (tBu)-D-Lys-Leu-Lys-Pro-D-Ala-NH-resin, N, N'-diethyl carbodiimide and Ytterbiumtriflate mol ratio are 1:2:1%, temperature of reaction: 25 ℃, the reaction times is 3 hours.
5. method according to claim 2, is characterized in that:
Wherein, described aminoresin is: Rink Amide-MBHA Resin resin, Rink Amide-BHA Resin resin, Rink Amide-AM Resin resin, Sieber Resin resin, a kind of in Sieber-AM Resin resin.
6. method according to claim 2, is characterized in that:
Wherein, described coupling agent system comprises condensing agent and reaction solvent, and described condensing agent is selected from DIC/HOBt, PyBOP/HOBt/DIEA or HATU/HOBt/DIEA; Described reaction solvent is selected from DMF, DCM, NMP or the combination between them.
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Cited By (3)
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|---|---|---|---|---|
| WO2017114414A1 (en) * | 2015-12-29 | 2017-07-06 | 深圳翰宇药业股份有限公司 | Method for detecting ganirelix acetate |
| CN107056894A (en) * | 2017-05-26 | 2017-08-18 | 济南康和医药科技有限公司 | A kind of method of fragment method synthesis in solid state ganirelix acetate |
| WO2023101490A1 (en) * | 2021-12-01 | 2023-06-08 | 애니젠 주식회사 | Novel method for manufacturing ganirelix |
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| US5212288A (en) * | 1990-02-20 | 1993-05-18 | Syntex (U.S.A.) Inc. | Temporary minimal protection synthesis of serine-containing polypeptides |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017114414A1 (en) * | 2015-12-29 | 2017-07-06 | 深圳翰宇药业股份有限公司 | Method for detecting ganirelix acetate |
| CN107056894A (en) * | 2017-05-26 | 2017-08-18 | 济南康和医药科技有限公司 | A kind of method of fragment method synthesis in solid state ganirelix acetate |
| WO2023101490A1 (en) * | 2021-12-01 | 2023-06-08 | 애니젠 주식회사 | Novel method for manufacturing ganirelix |
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|---|---|
| CN104017058B (en) | 2016-08-24 |
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