CN101284863A - Preparation method of solid phase synthesis cetrorelix - Google Patents
Preparation method of solid phase synthesis cetrorelix Download PDFInfo
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- CN101284863A CN101284863A CNA2008100434541A CN200810043454A CN101284863A CN 101284863 A CN101284863 A CN 101284863A CN A2008100434541 A CNA2008100434541 A CN A2008100434541A CN 200810043454 A CN200810043454 A CN 200810043454A CN 101284863 A CN101284863 A CN 101284863A
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- fluorenylmethyloxycarbonyl
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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
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Abstract
A method for realizing the solid phase synthesis of cetrorelix comprises the following steps that: Fmoc-Linker-MBHA-Resin is used as a starting raw material and is connected in turn with amino acid with Fmoc-protecting groups according to a solid phase synthesis method, so as to obtain protective decapeptide resin; meanwhile, the Fmoc-protecting groups are divested in turn; one sort of HBTU/HOBT or DIC/HOBT is used as condensing agent to carry out synthesized peptide, thereby obtaining protective decapeptide resin; acetylation reaction is carried out, and then protecting group side chain divesting and peptide cutting are carried out synchronously to obtain cetrorelix acetate which is separated and purified through C18 or C8 chromatographic column; finally, cetrorelix acetate acetate or trifluoroacetate is obtained after freeze drying.
Description
Technical field:
The present invention relates to the synthetic preparation method of cetrorelix of preparation method, especially solid-phase polypeptide of cetrorelix acetate (comprising acetate, trifluoroacetate and free peptide).
Background technology:
Chinese name: cetrorelix acetate
English name: Cetrorelix Acetate
Structural formula:
AC-3-(2-naphthyl)
-D-Ala-4-Chloro-D-Phe-3(3-Pyriclyl-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2
Molecular formula and molecular weight: C70H92N17O14CL 1430.1
Cetrorelix Acetate is a kind of gonadotropin releasing hormone, and being used for stimulates modulated ovary before the sterility treatment.The blocking-up of main treatment gonadotropin-releasing hormone receptor.
Lhrh antagonist Cetrorelix inquires into the mechanism that it suppresses the inner membrance growth of cancer cells to the influence of carcinoma of endometrium cell growth cycle and cycle associated protein
Belgium Solvay company of drugmaker announces that endometriosis medicine cetrorelix (Cetrorelix) of company's exploitation has entered III phase clinical stage, might be listing in 2009.
The Cetrotide molecular weight has only 1431, may be destroyed by ferment in the intestines and stomach, can not be oral, only can subcutaneous or intramuscular injection, effect rapidly, one hour Nei Keda serum-concentration peak 30ng/ml, half life is about one day.It approximately exceeds 20 times than GnRH itself to the bonding force (binding affinity) of GnRH acceptor, also exceeds twice even compare with buserelin in the fortifier.The shortcoming that in the past most induced one to denounce as for antagonist---disengage histamine (histamine release), cause allergy, lower greatly, find in the experiment will reach thousand times at least, half amount of histamine that antagonist disengages before just can discharging to the effective therapeutic dose of serum; Clinically, in the position of accepting injection, have the skin reaction that some rednesses are scratched where it itches occasionally, real more serious systemic anaphylaxis has only an example report so far, be an ovarian cancer sufferer after accepting continuous seven months heavy dose (10mg/day) injection, blood pressure reduces, and erythema appears in skin, cough, but very fast recovery is normal in 20 minutes.
Owing to itself be to belong to antagonist, cetrotide does not have the irritant reaction at use initial stage, and the effect that suppresses LH can manifest immediately, so can shorten duration of service clinically, needn't be as used as the fortifier in the past, promptly bring into use from the luteal phase of last one-period, can just bring into use moving closer to 72-120 hour of may ovulate a few days ago with the cycle, two kinds of using method are arranged at present: single dose injection (single doseschedule) and multidose injection (multiple dose schedule).Single dose is the 7th day that stimulates at the ovulation injection to method, adopts subcutaneous injection cetrotide 3mg one time; The multidose rule is two days ahead of time, injects low dose of cetrotide 0.25mg from the 5th day every twenty four hours, till injection HCG.Two kinds of methods can effectively suppress the rising (premature LH surge) of early onset luteotrophic hormone, avoid the too early luteinization of archiblast, influence the quality of ovum.
Another advantage of Cetrotide is to reduce the incidence of ovarian hyperstimulation, because this medicine belongs to GnRH antagonists, meeting antagonism GnRH on the pharmacology, FSH, the LH all functions, certainly comprise that the folliculus stimulatory function of FSH and the similar HCG of LH bring out the ovary ovulation function, and then the reduction overstimulation may, the ratio of OHSS can be forced down into 1-2% by present 5-6% according to report, significantly reduced its threat, also reduce the possibility that sufferer need be in hospital, made infertile treatment have more hommization.
Synthetic solid phase synthesis and synthetic two kinds of methods of liquid phase of being divided into of Cetrorelix; owing in the peptide sequence a plurality of special amino acid such as D-Cit are arranged; the 3-D-pyrazoleahtnine; 4-chloro-D-phenylalanine is segmental synthetic also relative difficult in liquid phase is synthetic; be difficult to purifying; add that sequence is also long; several fragment combination are height and the product purity that obtains is not easy to do; so the synthetic not too suitable Cetrorelix's of liquid phase method is synthetic; and solid phase synthesis has Boc method and Fmoc/tBu method; the Boc method is used the aminomethyl resin; carry out the cutting of resin at last with HF; so HF on industrial application is because severe toxicity is restricted; so we adopt comparative maturity and the synthetic Cetrorelix of Fmoc/tBu method fast, adopt minimum protection philosophy in synthetic, directly use Fmoc-D-Cit to synthesize polypeptide as raw material; avoid using the D-Cit raw material of expensive band side chain protected, obtained more satisfactory synthetic result.
Some abbreviations commonly used have following implication among the present invention;
Fmoc: fluorenylmethyloxycarbonyl
TBu: the tertiary butyl
HOBt:1-hydroxybenzene a pair of horses going side by side triazole
HBTU: benzotriazole-N, N, N ', N ' phosphoric acid-tetramethyl-urea hexafluoro ester
DIC:N, N-di-isopropyl carbodiimide
DIEA:N, the N-diisopropylethylamine
Leu: leucine
Tyr: tyrosine
Arg: arginine
The D-Cit:D-citrulline
Pro: proline(Pro)
Ser: Serine
The 3-Pyridyl-D-Ala:3-D-pyrazoleahtnine
4-Chloro-D-Phe:4-chloro-D-phenylalanine
Pbf:2,2,4,6,7-pentamethyl-Dihydrobenzofuranes-5-sulphonyl
Boc: tertbutyloxycarbonyl
DMF:N, dinethylformamide
Piperidine: hexahydropyridine
Fmoc-Rink-MBHA-resin: fluorenylmethyloxycarbonyl 4-toluene hydrogen polyimide resin
TFA: trifluoracetic acid
MeOH: methyl alcohol
CH
2CL
2: methylene dichloride
Kniser test detection reagent is: triketohydrindene hydrate
Summary of the invention:
The object of the present invention is to provide a kind of preparation method of cetrorelix, mainly solve the technical problem of the environmental pollution that cutting toxicity that existing solid-phase synthesis uses HF to carry out resin causes greatly.The present invention prepares the synthetic result of ideal fast with the principle of minimum protection.
Technical scheme of the present invention: a kind of preparation method of solid phase synthesis cetrorelix may further comprise the steps:
Make starting raw material with Fmoc-Linker-MBHA-Resin; method according to solid phase synthesis; connect amino acid successively with Fmoc-blocking group; obtain the decapeptide resin of protection; slough the Fmoc-blocking group therebetween successively; connect reactive polypeptide with a kind of among HBTU/HOBT or the DIC/HOBT for condensing agent; behind the decapeptide resin that must protect; carry out acetylization reaction; take off the side chain protected group then synchronously and cut peptide, obtain cetrorelix acetate, and carry out separation and purification through C18 or C8 chromatographic column; lyophilize again obtains cetrorelix acetate acetate or trifluoroacetate.
Adopt the amino acid of Fmoc protection to be successively: Fmoc-Pro-OH, Fmoc-Arg (pbf)-OH, Fmoc-Leu-OH; Fmoc-D-Cit-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-OH; Fmoc-3 (3-Pyridyl)-D-Ala-OH, Fmoc-4-Chloro-D-Phe-OH.
Working concentration 20%Piperidine DMF solution takes off Fmoc.
The temperature of reaction that connects reactive polypeptide is 35-50 ℃, and the reaction times is 0.5-2 hour.
Acetylize adds aceticanhydride in resin, pyridine, and the mixing solutions of methylene dichloride (1: 2: 2 volume ratio) reacted 2 hours.
Add be chilled in advance-10-20 ℃ cut peptide reagent (trifluoracetic acid/mercaptan/tri isopropyl silane/water=87.5/5/5/2.5 or trifluoracetic acid/thioanisole/p-cresol/mercaptan/water=86.5/1/5/2.5/5, volume ratio), cut reactive polypeptide 1.5-3 hour for 30-50 ℃, leach solvent, add ether sedimentation, the collecting precipitation thing, with the ether washing,, put in the moisture eliminator in room temperature, drying under reduced pressure 2-5 hour, obtain the cetrorelix acetate crude product.
The cetrorelix acetate crude product is dissolved in the acetonitrile/water solution, filters, filtrate is through C18 or C8 column purification, moving phase: phosphoric acid salt or trifluoroacetate: acetonitrile (0.1-100: the 100-0.1 volume ratio); The detection wavelength is 220nm; Follow the tracks of the needed effluent liquid of collection with liquid chromatograph, the sample peak desalts after merging, and freeze-drying obtains finished product.
The invention has the beneficial effects as follows: cetrorelix operational path of the present invention has following characteristics, possesses scale words productivity, process stabilizing, and the starting material convenient sources, with short production cycle, production cost is low, yield height, steady quality.Employing is cut peptide reagent (trifluoracetic acid/thioanisole/p-cresol/mercaptan/water) and is cut peptide, adds the method for ether sedimentation, avoids using poisonous reagents such as hydrogen fluoride, and the yield height, adopts the C18 post to carry out separation and purification, and total recovery is 20%.The Fmoc route of this process using gentleness, per step takes off Fomc and only uses 20% hexahydropyridine, cuts peptide and adopts a small amount of trifluoracetic acid, avoids using hydrogen fluoride, has reduced three waste discharge.
Embodiment:
Embodiment 1
(1) preparation Fmoc-D-Aal-resin
Fmoc-linker-MBHA-resin is soaked 2-5 time with DCM, make the abundant swelling of resin, add the reagent of raising one's hat, reaction, drain,, drain with the DMF washing, add the mixture that meets peptide reagent dissolved Fmoc-D-Ala-OH, HBTU/HOBT/DIEA, 35-50 ℃ connect reactive polypeptide 2 hours, drained, and resin washs with DMF, vacuum is drained, and obtains the Fmoc-D-Aal-resin.
The component of the said reagent of raising one's hat and volume ratio are 20%Piperidine DMF,
The mole number of Fmoc-D-Ala-OH is 2 times of resin, down together,
The weight of Fmoc-linker-MBHA-resin resin is the 8-15ml/g resin with the ratio of the add-on of the reagent of raising one's hat, down together,
The mole number of HBTU is 2 times of resin, down together.
The mole number of HOBT is 2 times of resin, down together.
The mole number of DIEA is 4 times of resin, down together.
(2) preparation Fmoc-Pro-D-Aal-resin
In Fmoc-D-Aal-resin 3 grams of step (1), add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat drained, respectively wash successively 3 times with DMF, MeOH, DCM, drain, add with connecing peptide reagent dissolved Fmoc-Pro-OH, the mixture of HBTU/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 2 hours, drain, drain, obtain the Fmoc-Pro-D-Aal-resin with the DMF washing.
The mole number of Fmoc-Pro-OH is 2 times of resin.
All the other operations and processing condition are with (1)
(3) preparation Fmoc-Arg (pbf)-Pro-D-Aal-resin
In the Fmoc-Pro-D-Aal-resin of step (2), add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat drained, respectively wash successively 3 times with DMF, MeOH, DCM, drain, add with connecing peptide reagent dissolved Fmoc-Arg (pbf)-OH, the mixture of HBTU/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 2 hours, drain, drain, obtain Fmoc-Arg (pbf)-Pro-D-Aal-resin with the DMF washing.
The mole number of Fmoc-Arg (pbf)-OH is 3 times of resin
All the other operations and processing condition are with (1).
(4) preparation Fmoc-Leu-Arg (pbf)-Pro-D-Aal-resin
In the Fmoc-Arg of step (3) (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat drained, respectively wash successively 3 times with DMF, MeOH, DCM, drain, add with connecing peptide reagent dissolved Fmoc-Leu-OH, the mixture of HBTU/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 2 hours, drain, drain, obtain Fmoc-Leu-Arg (pbf)-Pro-D-Aal-resin with the DMF washing.
The mole number of Fmoc-Leu-OH is 2 times of resin
All the other operations and processing condition are with (1).
(5) preparation Fmoc-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin
In the Fmoc-Leu-Arg of step (4) (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat drained, respectively wash successively 3 times with DMF, MeOH, DCM, drain, add with connecing peptide reagent dissolved Fmoc-D-Cit-OH, the mixture of HBTU/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 2 hours, drain, drain, obtain Fmoc-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin with the DMF washing.
The mole number of Fmoc-D-Cit-OH is 2 times of resin
All the other operations and processing condition are with (1).
(6) preparation Fmoc-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin
In the Fmoc-D-Cit-Leu-Arg of step (5) (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat drained, respectively wash successively 3 times with DMF, MeOH, DCM, drain, add with connecing peptide reagent dissolved Fmoc-Tyr (tBu)-OH, the mixture of HBTU/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 2 hours, drain, drain, obtain Fmoc-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin with the DMF washing.
The mole number of Fmoc-Tyr (tBu)-OH is 2 times of resin
All the other operations and processing condition are with (1).
(7) preparation Fmoc-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin is in the Fmoc-Tyr of step (6) (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat, drain, use DMF, MeOH, DCM respectively washs 3 times successively, drain, add with meeting peptide reagent dissolved Fmoc-Ser (tBu)-OH, the mixture of HBTU/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 2 hours, drain, drain, obtain Fmoc-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin with the DMF washing.
The mole number of Fmoc-Ser (tBu)-OH is 2 times of resin
All the other operations and processing condition are with (1).
(8) preparation Fmoc-3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin
In the Fmoc-Ser of step (7) (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat, drain, use DMF, MeOH, DCM respectively washs 3 times successively, drain, add with meeting peptide reagent dissolved Fmoc-3 (3-Pyridyl)-D-Ala-OH, the mixture of HBTU/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 2 hours, drain, drain, obtain Fmoc-3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin with the DMF washing.
The mole number of Fmoc--3 (3-Pyridyl)-D-Ala-OH is 2 times of resin
All the other operations and processing condition are with (1).
(9) preparation Fmoc-4-Chloro-D-Phe--3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin is in the Fmoc-3 of step (8) (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat, drain, use DMF, MeOH, DCM respectively washs 3 times successively, drain, add with meeting peptide reagent dissolved Fmoc-Chloro-D-Phe-OH, the mixture of HBTU/HOBT/DIEA, 30-50 ℃ connects reactive polypeptide 0.5-2 hour, drain, drain with the DMF washing, obtain Fmoc-Chloro-D-Phe--3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin.
The mole number of Fmoc-4-Chloro-D-Phe-OH is 2 times of resin
All the other operations and processing condition are with (1).
(10) preparation Fmoc-3-(2-naphthy1)-D-Ala-4-Chloro-D-Phe--3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin is in the Fmoc-4-Chloro-D-Phe-3 of step (9) (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat, drain, use DMF, MeOH, DCM respectively washs 3 times successively, drain, add with meeting peptide reagent dissolved Fmoc-3-(2-naphthyl)-D-Ala-OH, the mixture of HBTU/HOBT/DIEA, 30-50 ℃ connects reactive polypeptide 0.5-2 hour, drain, drain with the DMF washing, obtain Fmoc-3-(2-naphthy1)-D-Ala-4-Chloro-D-Phe--3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin.
The mole number of Fmoc-3-(2-naphthy1)-D-Ala-OH is 2 times of resin.
All the other operations and processing condition are with (1).
(11) preparation AC-3-(2-naphthy1)-D-Ala-4-Chloro-D-Phe-3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin is in the Fmoc-4-Chloro-D-Phe-3 of step (10) (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat, drain, use DMF, MeOH, DCM respectively washs 3 times successively, drain, add aceticanhydride, pyridine, the mixing solutions of methylene dichloride, 30-50 ℃ connect reactive polypeptide 2 hours, drain, drain with the DMF washing, obtain AC-3-(2-naphthy1)-D-Ala-4-Chloro-D-Phe--3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin.
Aceticanhydride, pyridine, the mixed liquor volume ratio of methylene dichloride is 1: 2: 2.
(12) preparation AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3 (3-Pyriclyl-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH
2
In the AC-4-Chloro-D-Phe--3 of step (11) (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin, add be chilled in advance-10-20 ℃ cut peptide reagent (trifluoracetic acid/mercaptan/tri isopropyl silane/water=87.5/5/5/2.5 or trifluoracetic acid/thioanisole/p-cresol/mercaptan/water=86.5/1/5/2.5/5, volume ratio), cut reactive polypeptide 1.5-3 hour for 30-50 ℃, leach solvent, add ether sedimentation, the collecting precipitation thing, wash with ether, in room temperature, put in the moisture eliminator, drying under reduced pressure 2-5 hour, obtaining cetrorelix acetate crude product 1.95 gram (91.0%) purity was 76.3%.
Embodiment 2
(1) preparation Fmoc-Pro-D-Aal-resin
In Fmoc-D-Aal-resin 1 gram of embodiment 1 step (1), add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat drained, respectively wash successively 3 times with DMF, MeOH, DCM, drain, add with connecing peptide reagent dissolved Fmoc-Pro-OH, the mixture of DIC/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 0.5 hour, drain, drain, obtain the Fmoc-Pro-D-Aal-resin with the DMF washing.
The component of the said reagent of raising one's hat and volume ratio are 20%Piperidine DMF,
The mole number of Fmoc-Pro-OH is 4 times of resin.
The mole number of DIC is 2 times of resin, down together.
The mole number of HOBT is 2 times of resin, down together.
The mole number of DIEA is 4 times of resin, down together.
(2) preparation Fmoc-Arg (pbf)-Pro-D-Aal-resin
In the Fmoc-Pro-D-Aal-resin of step (1), add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat drained, respectively wash successively 3 times with DMF, MeOH, DCM, drain, add with connecing peptide reagent dissolved Fmoc-Arg (pbf)-OH, the mixture of DIC/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 0.5 hour, drain, drain, obtain Fmoc-Arg (pbf)-Pro-D-Aal-resin with the DMF washing.
The mole number of Fmoc-Arg (pbf)-OH is 4 times of resin
All the other operations and processing condition are with (1).
(3) preparation Fmoc-Leu-Arg (pbf)-Pro-D-Aal-resin
In the Fmoc-Arg of step (2) (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat drained, respectively wash successively 3 times with DMF, MeOH, DCM, drain, add with connecing peptide reagent dissolved Fmoc-Leu-OH, the mixture of DIC/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 0.5 hour, drain, drain, obtain Fmoc-Leu-Arg (pbf)-Pro-D-Aal-resin with the DMF washing.
The mole number of Fmoc-Leu-OH is 4 times of resin
All the other operations and processing condition are with (1).
(4) preparation Fmoc-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin
In the Fmoc-Leu-Arg of step (4) (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat drained, respectively wash successively 3 times with DMF, MeOH, DCM, drain, add with connecing peptide reagent dissolved Fmoc-D-Cit-OH, the mixture of DIC/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 0.5 hour, drain, drain, obtain Fmoc-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin with the DMF washing.
The mole number of Fmoc-D-Cit-OH is 4 times of resin
All the other operations and processing condition are with (1).
(5) preparation Fmoc-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin
In the Fmoc-D-Cit-Leu-Arg of step (4) (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat drained, respectively wash successively 3 times with DMF, MeOH, DCM, drain, add with connecing peptide reagent dissolved Fmoc-Tyr (tBu)-OH, the mixture of DIC/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 0.5 hour, drain, drain, obtain Fmoc-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin with the DMF washing.
The mole number of Fmoc-Tyr (tBu)-OH is 4 times of resin
All the other operations and processing condition are with (1).
(6) preparation Fmoc-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin is in the Fmoc-Tyr of step (5) (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat, drain, use DMF, MeOH, DCM respectively washs 3 times successively, drain, add with meeting peptide reagent dissolved Fmoc-Ser (tBu)-OH, the mixture of DIC/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 0.5 hour, drain, drain, obtain Fmoc-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin with the DMF washing.
The mole number of Fmoc-Ser (tBu)-OH is 4 times of resin
All the other operations and processing condition are with (1).
(7) preparation Fmoc-3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin
In the Fmoc-Ser of step (8) (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat, drain, use DMF, MeOH, DCM respectively washs 3 times successively, drain, add with meeting peptide reagent dissolved Fmoc-3 (3-Pyridyl)-D-Ala-OH, the mixture of DIC/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 0.5 hour, drain, drain, obtain Fmoc-3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin with the DMF washing.
The mole number of Fmoc--3 (3-Pyridyl)-D-Ala-OH is 4 times of resin
All the other operations and processing condition are with (1).
(8) preparation Fmoc-4-Chloro-D-Phe-3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin is in the Fmoc-3 of step (7) (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat, drain, use DMF, MeOH, DCM respectively washs 3 times successively, drain, add with meeting peptide reagent dissolved Fmoc-Chloro-D-Phe-OH, the mixture of DIC/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 0.5 hour, drain, drain with the DMF washing, obtain Fmoc-Chloro-D-Phe--3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin.
The mole number of Fmoc-4-Chloro-D-Phe--OH is 4 times of resin
All the other operations and processing condition are with (1).
(9) preparation Fmoc-3-(2-naphthy1)-D-Ala-4-Chloro-D-Phe--3 (3-Pyridyl)-D-Ala-Ser (tBu)-
Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin
In the Fmoc-4-Chloro-D-Phe--3 of step (8) (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat, drain, use DMF, MeOH, DCM respectively washs 3 times successively, drain, add with meeting peptide reagent dissolved Fmoc-3-(2-naphthy1)-D-Ala-OH, the mixture of DIC/HOBT/DIEA, 30-50 ℃ connect reactive polypeptide 0.5 hour, drain, drain with the DMF washing, obtain Fmoc-3-(2-naphthy1)-D-Ala-4-Chloro-D-Phe--3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin.
The mole number of Fmoc-3-(2-naphthy1)-D-Ala-OH is 4 times of resin
All the other operations and processing condition are with (1).
(10) preparation AC-3-(2-naphthy1)-D-Ala-4-Chloro-D-Phe--3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin is in the Fmoc-4-Chloro-D-Phe--3 of step (9) (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin, add the reagent of raising one's hat, 35-50 ℃ of reaction 20-30 minute of raising one's hat, drain, use DMF, MeOH, DCM respectively washs 3 times successively, drain, add aceticanhydride, pyridine, the mixing solutions of methylene dichloride, 30-50 ℃ connect reactive polypeptide 2 hours, drain, drain with the DMF washing, obtain AC-3-(2-naphthy1)-D-Ala-4-Chloro-D-Phe--3 (3-Pyridyl)-D-Ala-Ser (tBu)-Tyr (tBu)-D-Cit-Leu-Arg (pbf)-Pro-D-Aal-resin.
Aceticanhydride, pyridine, the mixed liquor volume ratio of methylene dichloride is 1: 2: 2.
(11) preparation AC-3-(2-naphthyl)
-D-Ala-4-Chloro-D-Phe-3 (3-Pyriclyl-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH
2With the method for embodiment 1 (12), obtaining cetrorelix acetate crude product 0.55 gram (76.9%) purity is 63.5%.
Embodiment 3
According to the present invention, crude product comprises the steps: through the method for C18 (C8) column separating purification
The cetrorelix acetate crude product is dissolved in the acetonitrile/water solution, filters, filtrate is through C18 or C8 column purification, moving phase: phosphoric acid salt or trifluoroacetate: acetonitrile (0.1-100: the 100-0.1 volume ratio); The detection wavelength is 220nm; Follow the tracks of the needed effluent liquid of collection with liquid chromatograph, the sample peak desalts after merging, and freeze-drying obtains finished product, and total recovery is 20% (in the mmol of resin).The weight concentration of acetonitrile is 0.5-10%, and the weight concentration of cetrorelix acetate is 1-10% in the acetonitrile.
Claims (7)
1; a kind of preparation method of solid phase synthesis cetrorelix; it is characterized in that may further comprise the steps: make starting raw material with fluorenylmethyloxycarbonyl 4-toluene hydrogen polyimide resin; method according to solid phase synthesis; connect amino acid successively with fluorenylmethyloxycarbonyl protection; obtain the decapeptide resin of protection; slough fluorenylmethyloxycarbonyl therebetween successively; with benzotriazole-N; N; N '; N ' phosphoric acid-tetramethyl-urea hexafluoro ester/1-hydroxybenzene a pair of horses going side by side triazole or N; a kind of for condensing agent connects reactive polypeptide in N-di-isopropyl carbodiimide/1-hydroxybenzene a pair of horses going side by side triazole behind the decapeptide resin that must protect, carries out acetylization reaction; take off the side chain protected group then synchronously and cut peptide; obtain the cetrorelix acetate crude product, the cetrorelix acetate crude product is dissolved in the acetonitrile/water solution filters, and carry out separation and purification through C18 or C8 chromatographic column; lyophilize again obtains cetrorelix acetate acetate or cetrorelix acetate trifluoroacetate.
2, the preparation method of solid phase synthesis cetrorelix according to claim 1; it is characterized in that adopting successively the amino acid of fluorenylmethyloxycarbonyl protection to be: the fluorenylmethyloxycarbonyl proline(Pro); fluorenylmethyloxycarbonyl (2; 2; 4; 6; 7-pentamethyl-Dihydrobenzofuranes-5-sulphonyl) arginine; the fluorenylmethyloxycarbonyl leucine; fluorenylmethyloxycarbonyl-D-citrulline, fluorenylmethyloxycarbonyl (tertiary butyl) tyrosine, fluorenylmethyloxycarbonyl (tertiary butyl) Serine; fluorenylmethyloxycarbonyl-3-D-pyrazoleahtnine, fluorenylmethyloxycarbonyl-4-chloro-D-phenylalanine.
3, the preparation method of solid phase synthesis cetrorelix according to claim 1 is characterized in that sloughing the N of fluorenylmethyloxycarbonyl working concentration 20% hexahydropyridine, dinethylformamide solution.
4, the preparation method of solid phase synthesis cetrorelix according to claim 1, the temperature of reaction that it is characterized in that connecing reactive polypeptide is 35-50 ℃, the reaction times is 0.5-2 hour.
5, the preparation method of solid phase synthesis cetrorelix according to claim 1 is characterized in that described acetylization reaction, adds volume ratio and be the mixing solutions reaction 2 hours of 1: 2: 2 aceticanhydride, pyridine and methylene dichloride in the decapeptide resin.
6, the preparation method of solid phase synthesis cetrorelix according to claim 1, it is characterized in that the described peptide of cutting is: adding is chilled to-10～20 ℃ the peptide reagent of cutting in advance, the described peptide reagent of cutting is that volume ratio is trifluoracetic acid/mercaptan/tri isopropyl silane/water of 87.5/5/5/2.5 or trifluoracetic acid/thioanisole/p-cresol/mercaptan/water that volume ratio is 86.5/1/5/2.5/5, cut reactive polypeptide 1.5-3 hour for 30-50 ℃, leach solvent, add ether sedimentation, the collecting precipitation thing, wash with ether, in room temperature, put in the moisture eliminator, drying under reduced pressure 2-5 hour, obtain the cetrorelix acetate crude product.
7, the preparation method of solid phase synthesis cetrorelix according to claim 1 is characterized in that described C18 or C8 column separating purification, moving phase: phosphoric acid salt or trifluoroacetate: the volume ratio of acetonitrile is (0.1-100): (100-0.1); The detection wavelength is 220nm; Follow the tracks of the needed effluent liquid of collection with liquid chromatograph, the sample peak desalts after merging.
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| CN102423484A (en) * | 2011-11-23 | 2012-04-25 | 深圳翰宇药业股份有限公司 | Stable cetrorelix medicinal composition and preparation method thereof |
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