CN104177478B - A kind of method for synthesizing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 - Google Patents
A kind of method for synthesizing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 Download PDFInfo
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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
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The present invention relates to medical synthesis field, discloses a kind of method for synthesizing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.The amino resins that N-terminal coupling has the D alanine of protection group under condensation reagent and activating reagent effect and amino coupled has protection group is carried out esterification by the method for the invention, obtains peptide resin 1；According to the order of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 amino acid sequence C-terminal to N-terminal; from peptide resin 1; under condensation reagent and activating reagent effect; remaining protected amino acid is subjected to extension coupling one by one; every time corresponding peptide resin is obtained after extension coupling; final to obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin, then acidolysis obtains Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product, and Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purifying crude obtains Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 sterling.The present invention selects suitable synthetic schemes, selects adaptable amino resins and acid hydrolysis solution, is optimizing whole synthesis technique, considerably improving the purity of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, has higher total recovery, and to any environment non-hazardous.
Description
Technical field
The present invention relates to medical synthesis field, and in particular to a kind of method for synthesizing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
Background technology
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is that gonadotropin-releasing hormone (GRH) (GnRH) acceptor of Hui Ling pharmaceutical Co. Ltds of Denmark research and development suppresses
Agent class medicine, reversible inhibition hypophysis GnRH acceptors reduce the release that gonadotropin releasing hormone then suppresses testosterone.This product is led to
Suppression is crossed to the vital testosterone of prostate cancer continued propagation to delay the growth of prostate cancer and deterioration.Before hormone therapy
Row gland cancer but causes testosterone concentration to increase sharply to reduce the initial stage of testosterone concentration, and this initial impulse hormone receptor temporary can promote
Tumour growth rather than suppress it, and Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 then will not.U.S. FDA is main in December, 2008 approval Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 listing
Advanced prostate cancer patients are directed to, delay the disease of prostate cancer by suppressing testosterone.
III phase clinical studies show, the effect that Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 reduces testosterone concentration at least can be with Leuprorelin depot controlled release
Injection (Lupron Depot) compares favourably, and it is statistically significantly fast to reduce testosterone concentration.At the 3rd day for the treatment of, this
Product group 96% reaches the testosterone concentration of gonad, and Leuprorelin group effect is 0%.14th, this product group 99%, which reaches, gave birth to
The testosterone concentration of gland is grown, Leuprorelin group is 18%.
In clinical studies, prostate specific antigen (PSA) concentration can judge terminal as the 2nd curative effect of monitoring.Make
With PSA 64% is reduced after Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 2 weeks, 85% after January, 95% after March, suppress PSA all the time in whole 1 year for the treatment of.
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 structural formula is：
Ac-D-Nal-D-Cpa-D-Pal-Ser-Aph(Hor)-D-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Al a-
NH2
Report both at home and abroad on Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 preparation report is a lot, and United States Patent (USP) US5925730 uses Boc synthesis in solid state
Method, this method is small, only reports purity, product purity 98%, while this method and needs to use hydrofluoric acid (HF)
Cracking, there is larger harm to human and environment, is not suitable for large-scale industry synthesis, and its solid phase synthesis process can not be maximum
Degree improves the combined coefficient and quality of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
The content of the invention
In view of this, it is an object of the invention to provide a kind of method for synthesizing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 so that side of the present invention
Method improves the purity of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and has higher total recovery, Environmental Safety.
To achieve the above object, the present invention provides following technical scheme：
A kind of method for synthesizing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, comprises the following steps：
The D-alanine that step 1, N-terminal coupling have protection group has under condensation reagent and activating reagent effect with amino coupled
The amino resins of protection group carries out esterification, obtains peptide resin 1；
Step 2, the order according to Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 amino acid sequence C-terminal to N-terminal, from peptide resin 1, in condensation reagent and
Under activating reagent effect, remaining protected amino acid is subjected to extension one by one and is coupled, obtains corresponding peptide after extension coupling every time
Resin, final to obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin, then acidolysis obtains Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product, and Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purifying crude adds auspicious with obtaining
Gram sterling；
EDT that it is 1-10% for 80-95% TFA, percent by volume by percent by volume that the acidolysis, which uses, surplus are
The mixing acid hydrolysis solution acidolysis of water composition.
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 backbone amino acid has 10, and composition is as follows：
Ac-D-Nal1-D-Cpa2-D-Pal3-Ser4-Aph(Hor)5-D-Aph(Cbm)6-Leu7-Lys(iPr)8-Pro9-
D-Ala10-NH2。
Wherein, the amino of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 C-terminal is the amino being cleaved using lysate from amino resins, and it does not belong to
In the amino on amino acid.
The present invention for the synthesis technique that uses in the prior art easily cause Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 total recovery it is relatively low, to human and environment
There is the defects of endangering, select suitable synthetic schemes, and optimize whole synthesis technique, synthesis technique of the present invention both can be with
Using synthesis in solid state, liquid phase synthesis can also be used.
Protection group of the present invention be Amino acid synthesis field commonly use protected amino acid main chain and side chain on amino,
The blocking group of the group of the interference such as carboxyl synthesis, prevents amino, carboxyl etc. from being reacted during target product is prepared, raw
Into impurity, for the amino acid for needing to protect side chain in the present invention, its side-chain structure as well known to those skilled in the art and
Know using conventional protection group come groups such as the amino on protected amino acid side chain, carboxyls, preferably, the present invention passes through tBu
Protection group protects the side chain of serine；N is protected by Boc protection groups6The side chain of-(1- Methylethyls) lysine.In addition, at this
In the protected amino acid that invention methods described is related to, N-terminal is preferably protected by Fmoc protection groups.By protection group protection
Amino acid is referred to as protected amino acid.Preferably, remaining described protected amino acid be Fmoc-Pro, Fmoc-Lys (iPr, Boc),
Fmoc-Leu, Fmoc-D-Aph (Cbm), Fmoc-Aph (Hor), Fmoc-Ser (tBu), Fmoc-D-Pal, Fmoc-D-Cpa and
Ac-D-Nal。
Preferably, protection group described in step 1 is Fomc protection groups.
Preferably, the amino resins is Rink Amide AM resins, Rink Amide resins, RinkMBHA resins
Or Sieber resins.When preferably using Fomc protection amino resins amino, the structural formula of each resin is as follows, the ball table in left side
Show polystyrene resin：
Preferably, N-terminal coupling has a D-alanine of protection group and amino coupled has the amino resins of protection group
Mol ratio is 1-6:1, more preferably 2.5-3.5:1.
Preferably, the substitution value of the amino resins is 0.2-1.8mmol/g amino resins, more preferably 0.5-
1.0mmol/g amino resins.
Preferably, the condensation reagent is preferably N, and N- DICs (DIC), N, N- dicyclohexyls carbon two
Imines (DCC), hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus/organic base (PyBOP/ organic bases), 2- (7- nitrogen
Miscellaneous -1H- BTAs -1- bases) -1,1,3,3- tetramethylureas hexafluorophosphoric acid ester/organic base (HATU/ organic bases), benzo three
Nitrogen azoles-N, N, N', N'- tetramethylurea hexafluorophosphate/organic base (HBTU/ organic bases), O- BTAs-N, N, N', N'-
One kind in tetramethylurea tetrafluoro boric acid ester/organic base (TBTU/ organic bases).The mole dosage of the condensation reagent is preferably more
1~6 times of amino total mole number, more preferably 2.5~3.5 times in peptide resin.
It should be noted that the PyBOP/ organic bases, HATU/ organic bases, HBTU/ organic bases, TBTU/ organic bases,
Belong to the condensation reagent of four kinds of Dual systems in the present invention, i.e., PyBOP, HATU, HBTU need when in use respectively with organic base group
Used together into a kind of condensation reagent, wherein the organic base and PyBOP, HATU, HBTU, TBTU mol ratio are preferred
For for 1.3-3.0:1, more preferably 1.3-2:1.
Preferably, the organic base in the condensation reagent is preferably DIPEA (DIPEA), triethylamine
Or N- methylmorpholines (NMM), (TEA) more preferably DIPEA.
Preferably, the activating reagent is I-hydroxybenzotriazole (HOBt) or N- hydroxyl -7- azepine BTAs
(HOAt).The dosage of the activating reagent is preferably 1~6 times of amino total mole number in peptide resin, and more preferably 2.5~3.5
Times.
Preferably, the esterification and the reaction dissolvent of extension coupling use DMF.
Extension coupling of the present invention refers to that after first amino acid and amino resins coupling remaining amino acid is according to ground
Add the amino acid of the C-terminal of Rake amino acid to the order of N-terminal one by one with previous coupling that condensation reaction (backbone amino and carboxylic occurs
The condensation reaction of base) it is coupled.During present invention coupling, mole of the amino acid and corresponding peptide resin when extension is coupled every time
Than being preferably 1-6:1, more preferably 2.5-3.5:1；The coupling reaction time is preferably 60~300 minutes, more preferably 100
~140 minutes.Peptide resin corresponding to described refers to that D-Ala and amino resins are coupled the peptide resin 1 to be formed, Pro and peptide resin 1 occasionally
Join the peptide tree that peptide resin 3, Leu and the coupling of peptide resin 3 that the peptide resin 2 formed, Lys (iPr) and the coupling of peptide resin 2 are formed are formed
Fat 4, D-Aph (Cbm) and the coupling formation of peptide resin 4 peptide resin 5, Aph (Hor) and the peptide resin 6 of the coupling formation of peptide resin 5,
Peptide resin 8, D-Cpa and the peptide resin 8 that peptide resin 7, D-Pal and the coupling of peptide resin 7 that Ser and the coupling of peptide resin 6 are formed are formed
It is coupled the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin that the peptide resin 9 formed, Ac-D-Nal and the coupling of peptide resin 9 are formed.
In extension is coupled, because there is protection group at each amino acid N end, it is therefore desirable to it is even again first to remove N-terminal protection group
Connection, this is common knowledge for a person skilled in the art.The present invention preferably uses PIP/DMF (piperidines/N, N- dimethyl formyls
Amine) mixed solution removing N-terminal protection group containing piperidines is 10~30% (V) in mixed solution, remaining is DMF.Go N-terminal protection group
Time is preferably 10~60 minutes, preferably 15~25 minutes.The dosage for removing N-terminal protection group reagent is preferably per 0.05mol
Polypeptide resin 1000-1600mL.
It should be noted that peptide resin of the present invention refer to any number amino acid according to Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 amino acid sequence and
Amino resins is connected the peptide resin to be formed, and also includes peptide resin 1 among these.
Preferably, the acidolysis use by percent by volume for 90% TFA, percent by volume be 5% EDT, remaining
Measure as the mixing acid hydrolysis solution acidolysis of water composition.The mixing acid hydrolysis solution dosage be preferably every gram of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin need 4~
15mL, more preferably 9~11mL.The time of the acidolysis is preferably more preferably 3~4 hours 1~6 hour under room temperature condition.
Preferably, the purifying is specially：
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product, 0.1%TFA/ aqueous dissolutions, 0.45 μm of filtering with microporous membrane of solution, purifying are standby；
Purified using high performance liquid chromatography, purifying chromatograph packing material is 10 μm of anti-phase C18, and flow phase system is
The 0.1%TFA/ aqueous solution -0.1%TFA/ acetonitrile solutions, 77mm*250mm column flow rate is 90mL/min, using gradient system
System elution, circulation sample introduction purifying, takes crude product solution to be splined in chromatographic column, starts mobile phase elution, collects main peak and boils off acetonitrile
Afterwards, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purifying intermediate concentrate is obtained；
Take Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 to purify intermediate concentrate, filtered with 0.45 μm of filter membrane standby；
Change salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, purifying chromatogram
Filler is 10 μm of anti-phase C18, and 77mm*250mm column flow rate is 90mL/min, using gradient elution, sample prescription in circulation
Method, it is splined in chromatographic column, starts mobile phase elution, gather collection of illustrative plates, observe the change of trap, collection changes salt main peak and with dividing
Liquid phase detection purity is analysed, merging changes salt main peak solution, is concentrated under reduced pressure, obtains Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aqueous acetic acid, is freeze-dried, obtains ground
Add Rake sterling.
The Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 synthesized by the method for the invention detects through HPLC, and purity is more than 99%, maximum single contaminant
Only 0.10% or so, total recovery reaches as high as 55.9%, higher than 98% purity disclosed in US5925730, and does not adopt
With HF, Environmental Safety.
From above technical scheme, the present invention selects suitable synthetic schemes, selects adaptable amino resins and acid
Liquid is solved, whole synthesis technique is being optimized, is considerably improving the purity of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, there is higher total recovery, and it is right
Any environment non-hazardous.
Embodiment
The invention discloses a kind of method for synthesizing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, those skilled in the art can use for reference present disclosure, fit
When modified technique parameter is realized.In particular, all similar replacements and change for a person skilled in the art
It is it will be apparent that they are considered as being included in the present invention.The method of the present invention is retouched by preferred embodiment
State, related personnel substantially can not depart from present invention, in spirit and scope to compound as described herein and preparation side
Method is modified or suitably changed with combining, to realize and using the technology of the present invention.
In the specific embodiment of the invention, the amino acid in the present invention is purchased from the limited public affairs of Chengdu sunshine Rong's biotechnology
Department, resin used are purchased from Shangyu pul resin Co., Ltd, and Chinese implication corresponding to english abbreviation used is shown in application documents
Table 1.
The english abbreviation lexical or textual analysis of table 1
With reference to embodiment, the present invention is expanded on further.
Embodiment 1：The synthesis of peptide resin 1
0.15mol Fmoc-D-Ala and 0.15mol HOBt are taken, are dissolved with appropriate DMF；0.15mol DIC separately are taken, are stirred
Under be slowly added into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the protection ammonia after being activated
Base acid solution, it is standby.
0.05mol Fmoc-Rink Amide AM resins (substitution value about 0.6mmol/g) are taken, using 1000mL 20%
PIP/DMF solution deprotects 25 minutes, and washing and filtering obtains Fmoc resin.
Fmoc-D-Ala solution after activation is added to and gone in Fmoc resin, reaction 6 hours is stirred at room temperature, takes out
Reaction solution, after DMF is washed 3 times, DCM is washed 3 times, and methanol washs 3 times, and each wash time is 3min, obtains Fmoc-D-Ala-
Rink Amide AM resins, i.e. peptide resin 1.
Embodiment 2：The synthesis of peptide resin 1
0.15mol Fmoc-D-Ala and 0.15mol HOBt are taken, are dissolved with appropriate DMF；0.15mol DCC separately are taken, are stirred
Under be slowly added into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the protection ammonia after being activated
Base acid solution, it is standby.
0.05mol Fmoc-Rink Amide resins (substitution value about 0.8mmol/g) are taken, using 1600mL20%PIP/
DMF solution deprotects 25 minutes, and washing and filtering obtains Fmoc resin.
Fmoc-D-Ala solution after activation is added to and gone in Fmoc resin, reaction 6 hours is stirred at room temperature, takes out
Reaction solution, after DMF is washed 3 times, DCM is washed 3 times, and methanol washs 3 times, and each wash time is 3min, obtains Fmoc-D-Ala-
Rink Amide resins, i.e. peptide resin 1.
Embodiment 3：The synthesis of peptide resin 1
0.15mol Fmoc-D-Ala and 0.15mol HOBt are taken, are dissolved with appropriate DMF；0.15mol DIC separately are taken, are stirred
Under be slowly added into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the protection ammonia after being activated
Base acid solution, it is standby.
0.05mol Fmoc-Rink mbha resins (substitution value about 0.5mmol/g) are taken, using 1200mL20%PIP/
DMF solution deprotects 25 minutes, and washing and filtering obtains Fmoc resin.
Fmoc-D-Ala solution after activation is added to and gone in Fmoc resin, reaction 6 hours is stirred at room temperature, takes out
Reaction solution, after DMF is washed 3 times, DCM is washed 3 times, and methanol washs 3 times, and each wash time is 3min, obtains Fmoc-D-Ala-
Rink mbha resins, i.e. peptide resin 1.
Embodiment 4：The synthesis of peptide resin 1
0.15mol Fmoc-D-Ala and 0.15mol HOBt are taken, are dissolved with appropriate DMF；0.15mol DIC separately are taken, are stirred
Under be slowly added into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the protection ammonia after being activated
Base acid solution, it is standby.
0.05mol Fmoc-Sieber resins (substitution value about 1.0mmol/g) are taken, using 1500mL 20%PIP/DMF
Solution deprotects 25 minutes, and washing and filtering obtains Fmoc resin.
Fmoc-D-Ala solution after activation is added to and gone in Fmoc resin, reaction 6 hours is stirred at room temperature, takes out
Reaction solution, after DMF is washed 3 times, DCM is washed 3 times, and methanol washs 3 times, and each wash time is 3min, obtains Fmoc-D-Ala-
Sieber resins, i.e. peptide resin 1.
Embodiment 5：The synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin
0.1mol Fmoc-Pro and 0.1mol HOBt are taken, are dissolved with appropriate DMF；0.1mol DIC separately are taken, it is slow under stirring
Slow addition, stirring reaction 30 minutes in room temperature environment, the Freamine Ⅲ after being activated are standby；
The peptide resin 1 of 0.05mol embodiments 1 is taken, is deprotected 25 minutes using 1000mL 20%PIP/DMF solution, washing
It is filtrated to get Fmoc peptide resin 1；
Freamine Ⅲ after activation is added in Fmoc peptide resin 1, coupling reaction 60~300 minutes, reacted
Terminal is defined by ninhydrin method detection, filtration washing, obtains peptide resin 2, Fmoc-Pro-D-Ala-Rink Amide AM resins.
Be sequentially connected remaining amino acid according to the method for above-mentioned synthesis peptide resin 2 (Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 C sections to N-terminal order, often
Secondary connection is corresponding obtain peptide resin 3,4,5,6,7,8,9)：Fmoc-Lys(iPr,Boc)、Fmoc-Leu、Fmoc-D-Aph
(Cbm), Fmoc-Aph (Hor), Fmoc-Ser (tBu), Fmoc-D-Pal, Fmoc-D-Cpa and Ac-D-Nal, add with obtaining auspicious
Gram resin, Ac-D-Nal-D-Cpa-D-Pal-Ser (tBu)-Aph (Hor)-D-Aph (Cbm)-Leu-Lys (iPr, Boc)-
Pro-D-Ala-Rink Amide AM resins.
Embodiment 6：The synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin
0.2mol Fmoc-Pro and 0.2mol HOBt are taken, are dissolved with appropriate DMF；0.2mol DCC separately are taken, it is slow under stirring
Slow addition, stirring reaction 30 minutes in room temperature environment, the Freamine Ⅲ being filtrated to get after activation are standby；
The peptide resin 1 of 0.05mol embodiments 2 is taken, is deprotected 25 minutes using 1400mL 20%PIP/DMF solution, washing
It is filtrated to get Fmoc peptide resin 1；
Freamine Ⅲ after activation is added in Fmoc peptide resin 1, coupling reaction 60~300 minutes, reacted
Terminal is defined by ninhydrin method detection, filtration washing, obtains peptide resin 2, Fmoc-Pro-D-Ala-Rink Amide resins.
Be sequentially connected remaining amino acid according to the method for above-mentioned synthesis peptide resin 2 (Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 C sections to N-terminal order, often
Secondary connection is corresponding obtain peptide resin 3,4,5,6,7,8,9)：Fmoc-Lys(iPr,Boc)、Fmoc-Leu、Fmoc-D-Aph
(Cbm), Fmoc-Aph (Hor), Fmoc-Ser (tBu), Fmoc-D-Pal, Fmoc-D-Cpa and Ac-D-Nal, add with obtaining auspicious
Gram resin, Ac-D-Nal-D-Cpa-D-Pal-Ser (tBu)-Aph (Hor)-D-Aph (Cbm)-Leu-Lys (iPr, Boc)-
Pro-D-Ala-Rink Amide resins.
Embodiment 7：The synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin
0.15mol Fmoc-Pro and 0.15mol HOBt are taken, are dissolved with appropriate DMF；0.14mol PyBOP separately are taken, are stirred
Under be slowly added into, stirring reaction 30 minutes in room temperature environment, add 0.28mol DIPEA, mix, the ammonia after being activated
Base acid solution, it is standby；
The peptide resin 1 of 0.05mol embodiments 3 is taken, is deprotected 25 minutes using 1300mL 20%PIP/DMF solution, washing
It is filtrated to get Fmoc peptide resin 1；
Freamine Ⅲ after activation is added in Fmoc peptide resin 1, coupling reaction 60~300 minutes, reacted
Terminal is defined by ninhydrin method detection, filtration washing, obtains peptide resin 2, Fmoc-Pro-D-Ala-Rink mbha resins.
Be sequentially connected remaining amino acid according to the method for above-mentioned synthesis peptide resin 2 (Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 C sections to N-terminal order, often
Secondary connection is corresponding obtain peptide resin 3,4,5,6,7,8,9)：Fmoc-Lys(iPr,Boc)、Fmoc-Leu、Fmoc-D-Aph
(Cbm), Fmoc-Aph (Hor), Fmoc-Ser (tBu), Fmoc-D-Pal, Fmoc-D-Cpa and Ac-D-Nal, add with obtaining auspicious
Gram resin, Ac-D-Nal-D-Cpa-D-Pal-Ser (tBu)-Aph (Hor)-D-Aph (Cbm)-Leu-Lys (iPr, Boc)-
Pro-D-Ala-Rink mbha resins.
Embodiment 8：The synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin
0.3mol Fmoc-Pro and 0.3mol HOBt are taken, are dissolved with appropriate DMF；0.3mol DIC separately are taken, it is slow under stirring
Slow addition, stirring reaction 30 minutes in room temperature environment, the Freamine Ⅲ after being activated are standby；
The peptide resin 1 of 0.05mol embodiments 4 is taken, is deprotected 25 minutes using 1300mL 20%PIP/DMF solution, washing
It is filtrated to get Fmoc peptide resin 1；
Freamine Ⅲ after activation is added in Fmoc peptide resin 1, coupling reaction 60~300 minutes, reacted
Terminal is defined by ninhydrin method detection, filtration washing, obtains peptide resin 2, Fmoc-Pro-D-Ala-Sieber resins.
Be sequentially connected remaining amino acid according to the method for above-mentioned synthesis peptide resin 2 (Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 C sections to N-terminal order, often
Secondary connection is corresponding obtain peptide resin 3,4,5,6,7,8,9)：Fmoc-Lys(iPr,Boc)、Fmoc-Leu、Fmoc-D-Aph
(Cbm), Fmoc-Aph (Hor), Fmoc-Ser (tBu), Fmoc-D-Pal, Fmoc-D-Cpa and Ac-D-Nal, add with obtaining auspicious
Gram resin, Ac-D-Nal-D-Cpa-D-Pal-Ser (tBu)-Aph (Hor)-D-Aph (Cbm)-Leu-Lys (iPr, Boc)-
Pro-D-Ala-Sieber resins.
Embodiment 9：The preparation of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 peptide resin made from Example 5, adding TFA, percent by volume that percent by volume is 90% is
5% EDT, the mixing acid hydrolysis solution acidolysis that surplus is water composition (10mL/ grams of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin of mixing acid hydrolysis solution), stirring is equal
It is even, reaction 3 hours is stirred at room temperature, reactant mixture is filtered using sand core funnel, collects filtrate, and resin washs 3 with a small amount of TFA again
It is secondary, it is concentrated under reduced pressure after merging filtrate, adds absolute ether precipitation, then wash precipitation 3 times with absolute ether, drain, obtain off-white color powder
End is Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product, and crude product purity is 80.3%.
Embodiment 10：The preparation of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 peptide resin made from Example 6, adding TFA, percent by volume that percent by volume is 85% is
7.5% EDT, the mixing acid hydrolysis solution acidolysis that surplus is water composition (15mL/ grams of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin of mixing acid hydrolysis solution), stirring is equal
It is even, reaction 3 hours is stirred at room temperature, reactant mixture is filtered using sand core funnel, collects filtrate, and resin washs 3 with a small amount of TFA again
It is secondary, it is concentrated under reduced pressure after merging filtrate, adds absolute ether precipitation, then wash precipitation 3 times with absolute ether, drain to obtain off-white powder
As Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product, crude product purity are 75.6%.
Embodiment 11：The preparation of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 peptide resin made from Example 7, adding TFA, percent by volume that percent by volume is 80% is
10% EDT, the mixing acid hydrolysis solution acidolysis that surplus is water composition (4mL/ grams of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin of mixing acid hydrolysis solution), stirring is equal
It is even, reaction 3 hours is stirred at room temperature, reactant mixture is filtered using sand core funnel, collects filtrate, and resin washs 3 with a small amount of TFA again
It is secondary, it is concentrated under reduced pressure after merging filtrate, adds absolute ether precipitation, then wash precipitation 3 times with absolute ether, drain to obtain off-white powder
As Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product, crude product purity are 77.1%.
Embodiment 12：The preparation of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 peptide resin made from Example 8, adding TFA, percent by volume that percent by volume is 95% is
1% EDT, the mixing acid hydrolysis solution acidolysis that surplus is water composition (8mL/ grams of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin of mixing acid hydrolysis solution), stir,
Reaction 3 hours is stirred at room temperature, reactant mixture is filtered using sand core funnel, collects filtrate, and resin is washed 3 times with a small amount of TFA again,
Be concentrated under reduced pressure after merging filtrate, add absolute ether precipitation, then with absolute ether wash precipitation 3 times, drain off-white powder i.e.
For Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product, crude product purity is 82.6%.
Embodiment 13：Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purifying crude
The gained Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product of Example 9, dissolved with purifying mobile phase A, 0.45 μm of filtering with microporous membrane of solution,
Purify standby；
Purified using high performance liquid chromatography, purifying chromatograph packing material is 10 μm of anti-phase C18, and flow phase system is
The 0.1%TFA/ aqueous solution -0.1%TFA/ acetonitrile solutions, 77mm*250mm column flow rate is 90mL/min, using gradient system
System elution, circulation sample introduction purifying, takes crude product solution to be splined in chromatographic column, starts mobile phase elution, collects main peak and boils off acetonitrile
Afterwards, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purifying intermediate concentrate is obtained；
Take Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 to purify intermediate concentrate, filtered with 0.45 μm of filter membrane standby；
Change salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, purifying chromatogram
Filler is 10 μm of anti-phase C18, and 77mm*250mm column flow rate is 90mL/min, using gradient elution, sample prescription in circulation
Method, it is splined in chromatographic column, starts mobile phase elution, gather collection of illustrative plates, observe the change of trap, collection changes salt main peak and with dividing
Liquid phase detection purity is analysed, merging changes salt main peak solution, is concentrated under reduced pressure, obtains Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aqueous acetic acid, is freeze-dried, obtains ground
Add Rake sterling 44.5g, total recovery 54.6%, molecular weight：1633.5 purity：99.7%, maximum single contaminant 0.12%.
Embodiment 14：Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purifying crude
The gained Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product of Example 10, dissolved with purifying mobile phase A, 0.45 μm of miillpore filter mistake of solution
Filter, purifying are standby；
Purified using high performance liquid chromatography, purifying chromatograph packing material is 10 μm of anti-phase C18, and flow phase system is
The 0.1%TFA/ aqueous solution -0.1%TFA/ acetonitrile solutions, 77mm*250mm column flow rate is 90mL/min, using gradient system
System elution, circulation sample introduction purifying, takes crude product solution to be splined in chromatographic column, starts mobile phase elution, collects main peak and boils off acetonitrile
Afterwards, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purifying intermediate concentrate is obtained；
Take Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 to purify intermediate concentrate, filtered with 0.45 μm of filter membrane standby；
Change salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, purifying chromatogram
Filler is 10 μm of anti-phase C18, and 77mm*250mm column flow rate is 90mL/min, using gradient elution, sample prescription in circulation
Method, it is splined in chromatographic column, starts mobile phase elution, gather collection of illustrative plates, observe the change of trap, collection changes salt main peak and with dividing
Liquid phase detection purity is analysed, merging changes salt main peak solution, is concentrated under reduced pressure, obtains Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aqueous acetic acid, is freeze-dried, obtains ground
Add Rake sterling 39.8g, total recovery 48.9%, molecular weight：1633.2 purity：99.6%, maximum single contaminant 0.10%.
Embodiment 15：Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purifying crude
The gained Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product of Example 11, dissolved with purifying mobile phase A, 0.45 μm of miillpore filter mistake of solution
Filter, purifying are standby；
Purified using high performance liquid chromatography, purifying chromatograph packing material is 10 μm of anti-phase C18, and flow phase system is
The 0.1%TFA/ aqueous solution -0.1%TFA/ acetonitrile solutions, 77mm*250mm column flow rate is 90mL/min, using gradient system
System elution, circulation sample introduction purifying, takes crude product solution to be splined in chromatographic column, starts mobile phase elution, collects main peak and boils off acetonitrile
Afterwards, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purifying intermediate concentrate is obtained；
Take Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 to purify intermediate concentrate, filtered with 0.45 μm of filter membrane standby；
Change salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, purifying chromatogram
Filler is 10 μm of anti-phase C18, and 77mm*250mm column flow rate is 90mL/min, using gradient elution, sample prescription in circulation
Method, it is splined in chromatographic column, starts mobile phase elution, gather collection of illustrative plates, observe the change of trap, collection changes salt main peak and with dividing
Liquid phase detection purity is analysed, merging changes salt main peak solution, is concentrated under reduced pressure, obtains Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aqueous acetic acid, is freeze-dried, obtains ground
Add Rake sterling 41.2g, total recovery 50.5%, molecular weight：1633.6 purity：99.5%, maximum single contaminant 0.11%.
Embodiment 16：Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purifying crude
The gained Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product of Example 12, dissolved with purifying mobile phase A, 0.45 μm of miillpore filter mistake of solution
Filter, purifying are standby；
Purified using high performance liquid chromatography, purifying chromatograph packing material is 10 μm of anti-phase C18, and flow phase system is
The 0.1%TFA/ aqueous solution -0.1%TFA/ acetonitrile solutions, 77mm*250mm column flow rate is 90mL/min, using gradient system
System elution, circulation sample introduction purifying, takes crude product solution to be splined in chromatographic column, starts mobile phase elution, collects main peak and boils off acetonitrile
Afterwards, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purifying intermediate concentrate is obtained；
Take Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 to purify intermediate concentrate, filtered with 0.45 μm of filter membrane standby；
Change salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, purifying chromatogram
Filler is 10 μm of anti-phase C18, and 77mm*250mm column flow rate is 90mL/min, using gradient elution, sample prescription in circulation
Method, it is splined in chromatographic column, starts mobile phase elution, gather collection of illustrative plates, observe the change of trap, collection changes salt main peak and with dividing
Liquid phase detection purity is analysed, merging changes salt main peak solution, is concentrated under reduced pressure, obtains Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aqueous acetic acid, is freeze-dried, obtains ground
Add Rake sterling 45.6g, total recovery 55.9%, molecular weight：1633.2 purity：99.7%, maximum single contaminant 0.09%.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
- A kind of 1. method for synthesizing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, it is characterised in that comprise the following steps：Step 1, N-terminal coupling have the D-alanine of protection group under condensation reagent and activating reagent effect and amino coupled has protection The amino resins of base carries out esterification, obtains peptide resin 1；Step 2, the order according to Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 amino acid sequence C-terminal to N-terminal, from peptide resin 1, in condensation reagent and activation Under reagent effect, remaining protected amino acid is subjected to extension one by one and is coupled, obtains corresponding peptide resin after extension coupling every time, Final to obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin, then acidolysis obtains Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product, and it is pure that Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purifying crude obtains Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 Product；EDT that it is 1-10% for 80-95% TFA, percent by volume by percent by volume that the acidolysis, which uses, surplus are water group Into mixing acid hydrolysis solution acidolysis, the resin carrier be Rink Amide AM resins, Rink Amide resins, RinkMBHA trees Fat or Sieber resins.
- 2. method according to claim 1, it is characterised in that protection group described in step 1 is Fomc protection groups.
- 3. method according to claim 1, it is characterised in that the N-terminal coupling has the D-alanine of protection group and amino even The mol ratio for being associated with the amino resins of protection group is 1-6:1.
- 4. method according to claim 1, it is characterised in that the amino acid and corresponding peptide resin during extension coupling every time Mol ratio is 1-6:1.
- 5. method according to claim 1, it is characterised in that the condensation reagent is N, N- DICs, N, N- dicyclohexylcarbodiimides, hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus/organic base, 2- (7- azepines - 1H- BTA -1- bases) -1,1,3,3- tetramethylureas hexafluorophosphoric acid ester/organic base, BTA-N, N, N', N'- tetra- In MU hexafluorophosphate/organic base, O- BTAs-N, N, N', N'- tetramethylurea tetrafluoro boric acid ester/organic base It is a kind of.
- 6. method according to claim 5, it is characterised in that the organic base be DIPEA, triethylamine or N- methylmorpholines.
- 7. method according to claim 1, it is characterised in that the activating reagent be I-hydroxybenzotriazole or N- hydroxyls- 7- azepine BTAs.
- 8. method according to claim 1, it is characterised in that the esterification and the reaction dissolvent of extension coupling use DMF。
- 9. method according to claim 1, it is characterised in that remaining described protected amino acid is Fmoc-Pro, Fmoc-Lys (iPr,Boc)、Fmoc-Leu、Fmoc-D-Aph(Cbm)、Fmoc-Aph(Hor)、Fmoc-Ser(tBu)、Fmoc-D-Pal、 Fmoc-D-Cpa and Ac-D-Nal.
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| CN107778354B (en) * | 2016-08-25 | 2021-03-02 | 成都圣诺生物制药有限公司 | Method for synthesizing abarelix |
| CN106749542B (en) * | 2016-12-27 | 2020-09-08 | 杭州固拓生物科技有限公司 | Synthetic method of fusirelin |
| CN107344960A (en) * | 2017-06-29 | 2017-11-14 | 凯莱英医药集团（天津）股份有限公司 | The preparation method of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 |
| CN109575109B (en) * | 2018-12-27 | 2022-03-25 | 兰州大学 | Method for preparing degarelix by fragment condensation |
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