CN104177478A - Method for synthesizing degarelix - Google Patents
Method for synthesizing degarelix Download PDFInfo
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- CN104177478A CN104177478A CN201410427405.3A CN201410427405A CN104177478A CN 104177478 A CN104177478 A CN 104177478A CN 201410427405 A CN201410427405 A CN 201410427405A CN 104177478 A CN104177478 A CN 104177478A
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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
The invention relates to the field of synthesis of medicines and discloses a method for synthesizing degarelix. The method for synthesizing the degarelix comprises the following steps: enabling D-alanine with a protective group coupled with an N end to carry out esterification reaction with amino resin with a protective group coupled with an amino in the presence of a condensation reagent and an activation reagent to obtain peptide resin 1; extending and coupling other protective amino acids one by one by starting from the peptide resin 1 according to a sequence from C end to N end of the amino acid sequence of the degarelix in the presence of the condensation reagent and the activation reagent, to obtain corresponding peptide resins after extending and coupling each time and finally obtain degarelix resin, then carrying out acidolysis to obtain a degarelix crude product, and purifying the degarelix crude product to obtain a degarelix pure product. By virtue of the method for synthesizing the degarelix, a proper synthesizing scheme is selected; the adaptive amino resin and an acidolysis solution are selected; the overall synthesis process is optimized; the purity of the degarelix is obviously improved; the degarelix has a relatively high total yield and is free of pollution to any environment.
Description
Technical field
The present invention relates to the synthetic field of medicine, be specifically related to a kind of method of synthetic 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 gonadotropin releasing hormone (GnRH) the acceptor inhibitor class medicine of Hui Ling pharmaceutical Co. Ltd of Denmark research and development, and reversible inhibition hypophysis GnRH acceptor reduces the release that gonadotropin releasing hormone suppresses testosterone then.This product is by suppressing the vital testosterone of the lasting growth of prostate cancer to delay growth and the deterioration of prostate cancer.The initial stage of reducing testosterone concentration with hormonotherapy prostate cancer but causes testosterone concentration to increase sharply, and this hormone receptor of this initial impulse can temporary promotion tumor growth instead of suppressed it, and Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 can not.U.S. FDA is ratified Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 listing in December, 2008, mainly for advanced prostate cancer patient, delays 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 compare favourably with Leuprolide depot controlled release injection (Lupron Depot), and it is significantly fast statistically to reduce testosterone concentration.At the 3rd day for the treatment of, this product group 96% reached gonadal testosterone concentration, and Leuprolide group effect is 0%.The 14th, this product group 99% reached gonadal testosterone concentration, and Leuprolide group is 18%.
In clinical study, the 2nd curative effect that prostate specific antigen (PSA) concentration can be used as monitoring judges terminal.After using Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 2 weeks, reduce PSA 64%, after January 85%, after March 95%, in whole 1 year that treats, suppress all the time PSA.
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-NH
2
A lot of about the report of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 preparation report both at home and abroad, US Patent No. 5925730 adopts Boc solid phase synthesis process, the method is small, only report purity, product purity is 98%, and the method need to be used hydrofluoric acid (HF) cracking simultaneously, and human and environment is had to larger harm, be not suitable for large-scale industry synthetic, and its solid phase synthesis process can not at utmost improve combined coefficient and the quality of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of method of synthetic Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, make the method for the invention improve the purity of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and there is higher total recovery, Environmental Safety.
For achieving the above object, the invention provides following technical scheme:
A method for synthetic 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 the coupling of step 1, N end has a protecting group under condensation reagent and activating reagent effect and amino coupled have the aminoresin of protecting group to carry out esterification, obtain peptide resin 1;
Step 2, the order of holding N end according to Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aminoacid sequence C, from peptide resin 1, under condensation reagent and activating reagent effect, all the other protected amino acids are extended to coupling one by one, after each extension coupling, all obtain corresponding peptide resin, the final Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin that obtains, 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 TFA that it is 80-95% by volume percent that described acidolysis adopts, EDT, the surplus that volume percent is 1-10% are the mixing acid hydrolysis solution acidolysis that water forms.
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 main chain amino acid has 10, composed as follows:
Ac-D-Nal
1-D-Cpa
2-D-Pal
3-Ser
4-Aph(Hor)
5-D-Aph(Cbm)
6-Leu
7-Lys(iPr)
8-Pro
9-D-Ala
10-NH
2。
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 end is to adopt the lysate amino that cracking is got off from aminoresin, and it does not belong to the amino on amino acid.
The synthesis technique that the present invention is directed to available technology adopting easily causes the defect that Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 total recovery is lower, human and environment is had to harm, select suitable synthetic schemes, and optimize whole synthesis technique, synthesis technique of the present invention both can adopt solid phase synthesis, also can adopt liquid phase synthetic.
Protecting group of the present invention is the blocking group of the synthetic group of the interference such as amino, carboxyl on the protected amino acid main chain conventional in the synthetic field of amino acid and side chain, prevent that amino, carboxyl etc. from reacting preparing in target product process, generate impurity, for the amino acid of the side chain that needs protection in the present invention, its side-chain structure as well known to those skilled in the art and know and adopt conventional protecting group to carry out the group such as amino, carboxyl on protected amino acid side chain, as preferably, the present invention is by the side chain of tBu protecting group protection Serine; By Boc protecting group protection N
6the side chain of-(1-methylethyl) Methionin.In addition,, in the protected amino acid relating in the method for the invention, N end is all preferably protected by Fmoc protecting group.The amino acid of protected base protection is called protected amino acid.As preferably; described all the other protected amino acids are 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.
As preferably, protecting group is Fomc protecting group described in step 1.
As preferably, described aminoresin is Rink Amide AM resin, Rink Amide resin, RinkMBHA resin or Sieber resin.In the time preferably adopting Fomc protection aminoresin amino, the structural formula of each resin is as follows, and the ball in left side represents polystyrene resin:
As preferably, it is 1-6:1, more preferably 2.5-3.5:1 that the coupling of described N end has the D-alanine of protecting group and amino coupled to have the mol ratio of the aminoresin of protecting group.
As preferably, the substitution value of described aminoresin is 0.2-1.8mmol/g aminoresin, more preferably 0.5-1.0mmol/g aminoresin.
As preferably, described condensation reagent is preferably N, N-DIC (DIC), N, N-dicyclohexylcarbodiimide (DCC), phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus/organic bases (PyBOP/ organic bases), 2-(7-azepine-1H-benzotriazole-1-yl)-1, 1, 3, 3-tetramethyl-urea phosphofluoric acid ester/organic bases (HATU/ organic bases), benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate/organic bases (HBTU/ organic bases), O-benzotriazole-N, N, N', one in N'-tetramethyl-urea Tetrafluoroboric acid ester/organic bases (TBTU/ organic bases).The mole dosage of described condensation reagent is preferably 1～6 times of amino total mole number in polypeptide resin, more preferably 2.5～3.5 times.
It should be noted that, described PyBOP/ organic bases, HATU/ organic bases, HBTU/ organic bases, TBTU/ organic bases, belong in the present invention the condensation reagent of four kinds of binary systems, be that PyBOP, HATU, HBTU need to combine and become a kind of condensation reagent use with organic bases respectively in use, the mol ratio of wherein said organic bases and PyBOP, HATU, HBTU, TBTU is preferably as 1.3-3.0:1, more preferably 1.3-2:1.
As preferably, the organic bases in described condensation reagent is preferably DIPEA (DIPEA), triethylamine (TEA) or N-methylmorpholine (NMM), more preferably DIPEA.
As preferably, described activating reagent is I-hydroxybenzotriazole (HOBt) or N-hydroxyl-7-azepine benzotriazole (HOAt).The consumption of described activating reagent is preferably 1～6 times of amino total mole number in peptide resin, more preferably 2.5～3.5 times.
As preferably, described esterification and the reaction solvent that extends coupling all adopt DMF.
Extension coupling of the present invention refers to after first amino acid and aminoresin coupling, and the order that remaining amino acid is held N end according to the amino acid whose C of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is carried out coupling with the amino acid generation condensation reaction of previous coupling (condensation reaction of the amino and carboxyl of main chain) one by one.When coupling of the present invention, while extending coupling at every turn described in the mol ratio of amino acid and corresponding peptide resin be preferably 1-6:1, more preferably 2.5-3.5:1; The described linked reaction time is preferably 60～300 minutes, more preferably 100～140 minutes.The peptide resin of described correspondence refers to the peptide resin 1 that D-Ala and aminoresin coupling form, the peptide resin 2 that Pro and peptide resin 1 coupling form, the peptide resin 3 that Lys (iPr) and peptide resin 2 couplings form, the peptide resin 4 that Leu and peptide resin 3 couplings form, the peptide resin 5 that D-Aph (Cbm) and peptide resin 4 couplings form, the peptide resin 6 that Aph (Hor) and peptide resin 5 couplings form, the peptide resin 7 that Ser and peptide resin 6 couplings form, the peptide resin 8 that D-Pal and peptide resin 7 couplings form, the peptide resin 9 that D-Cpa and peptide resin 8 couplings form, the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin that Ac-D-Nal and peptide resin 9 couplings form.
Extending in coupling, because each amino acid N end has protecting group, therefore need first to remove the coupling again of N end protecting group, this is common practise for a person skilled in the art.The present invention preferably uses PIP/DMF (piperidines/DMF) mixing solutions to remove N end protecting group, in mixing solutions, is 10～30% (V) containing piperidines, and all the other are DMF.Go the N end protecting group time to be preferably 10～60 minutes, preferably 15～25 minutes.Go the consumption of N end protecting group reagent to be preferably every 0.05mol polypeptide resin 1000-1600mL.
It should be noted that, peptide resin of the present invention refer to any number amino acid be connected with aminoresin 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 form peptide resin, this wherein also comprises peptide resin 1.
As preferably, the TFA that it is 90% by volume percent that described acidolysis adopts, EDT, the surplus that volume percent is 5% are the mixing acid hydrolysis solution acidolysis that water forms.Described mixing acid hydrolysis solution consumption is 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 needs 4～15mL, more preferably 9～11mL.The time of described acidolysis is preferably under room temperature condition 1～6 hour, more preferably 3～4 hours.
As preferably, described 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, the 0.1%TFA/ aqueous solution dissolves, 0.45 μ m filtering with microporous membrane for solution, purifying is for subsequent use;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purify intermediates concentrated solution;
Get Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purify intermediates concentrated solution, for subsequent use with 0.45 μ m filter membrane filtration;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopt gradient elution, quadrat method in circulation, is splined in chromatographic column, starts moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collects and changes salt main peak and with analyzing Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aqueous acetic acid, lyophilize, obtains Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 sterling.
The Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 being synthesized by the method for the invention detects through HPLC, and purity is more than 99%, and maximum single contaminant is only 0.10% left and right, and total recovery reaches as high as 55.9%, higher than disclosed 98% purity in US5925730, and does not adopt HF, Environmental Safety.
From above technical scheme, the present invention selects suitable synthetic schemes, selects the aminoresin and the acid hydrolysis solution that adapt, optimizing whole synthesis technique, improved significantly 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 to any environment without harm.
Embodiment
The invention discloses a kind of method of synthetic 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 content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, related personnel obviously can, not departing from content of the present invention, spirit and scope compound as herein described and preparation method changes or suitably change and combination, realize and apply the technology of the present invention.
In the specific embodiment of the invention, the amino acid in the present invention is purchased from Hui Rong bio tech ltd, Chengdu, and resin used is purchased from Shangyu pul resin company limited, and the Chinese implication that in application documents, english abbreviation used is corresponding is in table 1.
The lexical or textual analysis of table 1 english abbreviation
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: peptide resin 1 synthetic
Get 0.15mol Fmoc-D-Ala and 0.15mol HOBt, with appropriate DMF dissolving; Separately get 0.15mol DIC, be slowly added in protected amino acid DMF solution under stirring, stirring reaction 30 minutes in room temperature environment, obtains the protected amino acid solution after activation, for subsequent use.
Get the Fmoc-Rink Amide AM resin (the about 0.6mmol/g of substitution value) of 0.05mol, adopt 1000mL 20%PIP/DMF solution to go to protect 25 minutes, washing and filtering obtains the resin of Fmoc.
Fmoc-D-Ala solution after activation is joined in the resin that removes Fmoc, and stirring at room temperature reaction 6 hours, takes out reaction solution, after DMF washing 3 times, DCM washing 3 times, methanol wash 3 times, each washing time is 3min, obtains Fmoc-D-Ala-Rink Amide AM resin, i.e. peptide resin 1.
Embodiment 2: peptide resin 1 synthetic
Get 0.15mol Fmoc-D-Ala and 0.15mol HOBt, with appropriate DMF dissolving; Separately get 0.15mol DCC, be slowly added in protected amino acid DMF solution under stirring, stirring reaction 30 minutes in room temperature environment, obtains the protected amino acid solution after activation, for subsequent use.
Get the Fmoc-Rink Amide resin (the about 0.8mmol/g of substitution value) of 0.05mol, adopt 1600mL20%PIP/DMF solution to go to protect 25 minutes, washing and filtering obtains the resin of Fmoc.
Fmoc-D-Ala solution after activation is joined in the resin that removes Fmoc, and stirring at room temperature reaction 6 hours, takes out reaction solution, after DMF washing 3 times, DCM washing 3 times, methanol wash 3 times, each washing time is 3min, obtains Fmoc-D-Ala-Rink Amide resin, i.e. peptide resin 1.
Embodiment 3: peptide resin 1 synthetic
Get 0.15mol Fmoc-D-Ala and 0.15mol HOBt, with appropriate DMF dissolving; Separately get 0.15mol DIC, be slowly added in protected amino acid DMF solution under stirring, stirring reaction 30 minutes in room temperature environment, obtains the protected amino acid solution after activation, for subsequent use.
Get the Fmoc-Rink mbha resin (the about 0.5mmol/g of substitution value) of 0.05mol, adopt 1200mL20%PIP/DMF solution to go to protect 25 minutes, washing and filtering obtains the resin of Fmoc.
Fmoc-D-Ala solution after activation is joined in the resin that removes Fmoc, and stirring at room temperature reaction 6 hours, takes out reaction solution, after DMF washing 3 times, DCM washing 3 times, methanol wash 3 times, each washing time is 3min, obtains Fmoc-D-Ala-Rink mbha resin, i.e. peptide resin 1.
Embodiment 4: peptide resin 1 synthetic
Get 0.15mol Fmoc-D-Ala and 0.15mol HOBt, with appropriate DMF dissolving; Separately get 0.15mol DIC, be slowly added in protected amino acid DMF solution under stirring, stirring reaction 30 minutes in room temperature environment, obtains the protected amino acid solution after activation, for subsequent use.
Get the Fmoc-Sieber resin (the about 1.0mmol/g of substitution value) of 0.05mol, adopt 1500mL 20%PIP/DMF solution to go to protect 25 minutes, washing and filtering obtains the resin of Fmoc.
Fmoc-D-Ala solution after activation is joined in the resin that removes Fmoc, and stirring at room temperature reaction 6 hours, takes out reaction solution, after DMF washing 3 times, DCM washing 3 times, methanol wash 3 times, each washing time is 3min, obtains Fmoc-D-Ala-Sieber resin, i.e. peptide resin 1.
Embodiment 5: Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin synthetic
Get 0.1mol Fmoc-Pro and 0.1mol HOBt, with appropriate DMF dissolving; Separately get 0.1mol DIC, under stirring, slowly add, stirring reaction 30 minutes in room temperature environment, obtains the amino acid solution after activation, for subsequent use;
Get the peptide resin 1 of 0.05mol embodiment 1, adopt 1000mL 20%PIP/DMF solution to go to protect 25 minutes, washing and filtering obtains the peptide resin 1 of Fmoc;
Amino acid solution after activation is joined in the peptide resin 1 of Fmoc, linked reaction 60～300 minutes, reaction end detects and is as the criterion with ninhydrin method, and filtration washing, obtains peptide resin 2, Fmoc-Pro-D-Ala-Rink Amide AM resin.
According to the method for above-mentioned synthetic peptide resin 2, remaining amino acid is connected successively to (Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 C section is to N end order, each correspondence that connects obtains 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, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 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 resin.
Embodiment 6: Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin synthetic
Get 0.2mol Fmoc-Pro and 0.2mol HOBt, with appropriate DMF dissolving; Separately get 0.2mol DCC, under stirring, slowly add, stirring reaction 30 minutes in room temperature environment, filters the amino acid solution obtaining after activation, for subsequent use;
Get the peptide resin 1 of 0.05mol embodiment 2, adopt 1400mL 20%PIP/DMF solution to go to protect 25 minutes, washing and filtering obtains the peptide resin 1 of Fmoc;
Amino acid solution after activation is joined in the peptide resin 1 of Fmoc, linked reaction 60～300 minutes, reaction end detects and is as the criterion with ninhydrin method, and filtration washing, obtains peptide resin 2, Fmoc-Pro-D-Ala-Rink Amide resin.
According to the method for above-mentioned synthetic peptide resin 2, remaining amino acid is connected successively to (Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 C section is to N end order, each correspondence that connects obtains 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, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin, Ac-D-Nal-D-Cpa-D-Pal-Ser (tBu)-Aph (Hor)-D-Aph (Cbm)-Leu-Lys (iPr, Boc)-Pro-D-Ala-Rink Amide resin.
Embodiment 7: Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin synthetic
Get 0.15mol Fmoc-Pro and 0.15mol HOBt, with appropriate DMF dissolving; Separately get 0.14mol PyBOP, under stirring, slowly add, stirring reaction 30 minutes in room temperature environment, then add 0.28mol DIPEA, and mix, obtain the amino acid solution after activation, for subsequent use;
Get the peptide resin 1 of 0.05mol embodiment 3, adopt 1300mL 20%PIP/DMF solution to go to protect 25 minutes, washing and filtering obtains the peptide resin 1 of Fmoc;
Amino acid solution after activation is joined in the peptide resin 1 of Fmoc, linked reaction 60～300 minutes, reaction end detects and is as the criterion with ninhydrin method, and filtration washing, obtains peptide resin 2, Fmoc-Pro-D-Ala-Rink mbha resin.
According to the method for above-mentioned synthetic peptide resin 2, remaining amino acid is connected successively to (Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 C section is to N end order, each correspondence that connects obtains 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, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin, Ac-D-Nal-D-Cpa-D-Pal-Ser (tBu)-Aph (Hor)-D-Aph (Cbm)-Leu-Lys (iPr, Boc)-Pro-D-Ala-Rink mbha resin.
Embodiment 8: Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin synthetic
Get 0.3mol Fmoc-Pro and 0.3mol HOBt, with appropriate DMF dissolving; Separately get 0.3mol DIC, under stirring, slowly add, stirring reaction 30 minutes in room temperature environment, obtains the amino acid solution after activation, for subsequent use;
Get the peptide resin 1 of 0.05mol embodiment 4, adopt 1300mL 20%PIP/DMF solution to go to protect 25 minutes, washing and filtering obtains the peptide resin 1 of Fmoc;
Amino acid solution after activation is joined in the peptide resin 1 of Fmoc, linked reaction 60～300 minutes, reaction end detects and is as the criterion with ninhydrin method, and filtration washing, obtains peptide resin 2, Fmoc-Pro-D-Ala-Sieber resin.
According to the method for above-mentioned synthetic peptide resin 2, remaining amino acid is connected successively to (Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 C section is to N end order, each correspondence that connects obtains 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, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin, Ac-D-Nal-D-Cpa-D-Pal-Ser (tBu)-Aph (Hor)-D-Aph (Cbm)-Leu-Lys (iPr, Boc)-Pro-D-Ala-Sieber resin.
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
Get the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 peptide resin that embodiment 5 makes, adding volume percent is 90% TFA, volume percent is 5% EDT, surplus is the mixing acid hydrolysis solution acidolysis (mixing 10mL/ gram of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin of acid hydrolysis solution) of water composition, stir, stirring at room temperature reaction 3 hours, reaction mixture uses sand core funnel to filter, collect filtrate, resin is again with a small amount of TFA washing 3 times, concentrating under reduced pressure after merging filtrate, add anhydrous diethyl ether precipitation, wash precipitation 3 times with anhydrous diethyl ether again, drain, obtain off-white powder and be 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 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
Get the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 peptide resin that embodiment 6 makes, adding volume percent is 85% TFA, volume percent is 7.5% EDT, surplus is the mixing acid hydrolysis solution acidolysis (mixing 15mL/ gram of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin of acid hydrolysis solution) of water composition, stir, stirring at room temperature reaction 3 hours, reaction mixture uses sand core funnel to filter, collect filtrate, resin is again with a small amount of TFA washing 3 times, concentrating under reduced pressure after merging filtrate, add anhydrous diethyl ether precipitation, wash precipitation 3 times with anhydrous diethyl ether again, drain to such an extent that off-white powder is 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 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
Get the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 peptide resin that embodiment 7 makes, adding volume percent is that 80% TFA, EDT, the surplus that volume percent is 10% are the mixing acid hydrolysis solution acidolysis (mixing 4mL/ gram of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin of acid hydrolysis solution) of water composition, stir, stirring at room temperature reaction 3 hours, reaction mixture uses sand core funnel to filter, collect filtrate, resin is again with a small amount of TFA washing 3 times, concentrating under reduced pressure after merging filtrate, add anhydrous diethyl ether precipitation, wash precipitation 3 times with anhydrous diethyl ether again, drain to such an extent that off-white powder is 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 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
Get the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 peptide resin that embodiment 8 makes, adding volume percent is that 95% TFA, EDT, the surplus that volume percent is 1% are the mixing acid hydrolysis solution acidolysis (mixing 8mL/ gram of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin of acid hydrolysis solution) of water composition, stir, stirring at room temperature reaction 3 hours, reaction mixture uses sand core funnel to filter, collect filtrate, resin is again with a small amount of TFA washing 3 times, concentrating under reduced pressure after merging filtrate, add anhydrous diethyl ether precipitation, wash precipitation 3 times with anhydrous diethyl ether again, drain to such an extent that off-white powder is 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
Get embodiment 9 gained Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude products, with the dissolving of purifying mobile phase A, 0.45 μ m filtering with microporous membrane for solution, purifying is for subsequent use;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purify intermediates concentrated solution;
Get Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purify intermediates concentrated solution, for subsequent use with 0.45 μ m filter membrane filtration;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopt gradient elution, quadrat method in circulation, be splined in chromatographic column, start moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed salt main peak and is used and analyze Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aqueous acetic acid, lyophilize, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 sterling 44.5g, total recovery is 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
Get embodiment 10 gained Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude products, with the dissolving of purifying mobile phase A, 0.45 μ m filtering with microporous membrane for solution, purifying is for subsequent use;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purify intermediates concentrated solution;
Get Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purify intermediates concentrated solution, for subsequent use with 0.45 μ m filter membrane filtration;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopt gradient elution, quadrat method in circulation, be splined in chromatographic column, start moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed salt main peak and is used and analyze Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aqueous acetic acid, lyophilize, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 sterling 39.8g, total recovery is 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
Get embodiment 11 gained Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude products, with the dissolving of purifying mobile phase A, 0.45 μ m filtering with microporous membrane for solution, purifying is for subsequent use;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purify intermediates concentrated solution;
Get Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purify intermediates concentrated solution, for subsequent use with 0.45 μ m filter membrane filtration;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopt gradient elution, quadrat method in circulation, be splined in chromatographic column, start moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed salt main peak and is used and analyze Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aqueous acetic acid, lyophilize, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 sterling 41.2g, total recovery is 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
Get embodiment 12 gained Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude products, with the dissolving of purifying mobile phase A, 0.45 μ m filtering with microporous membrane for solution, purifying is for subsequent use;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purify intermediates concentrated solution;
Get Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 purify intermediates concentrated solution, for subsequent use with 0.45 μ m filter membrane filtration;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopt gradient elution, quadrat method in circulation, be splined in chromatographic column, start moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed salt main peak and is used and analyze Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aqueous acetic acid, lyophilize, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 sterling 45.6g, total recovery is 55.9%, molecular weight: 1633.2, purity: 99.7%, maximum single contaminant 0.09%.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a method for synthetic Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, is characterized in that, comprises the following steps:
The D-alanine that the coupling of step 1, N end has a protecting group under condensation reagent and activating reagent effect and amino coupled have the aminoresin of protecting group to carry out esterification, obtain peptide resin 1;
Step 2, the order of holding N end according to Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aminoacid sequence C, from peptide resin 1, under condensation reagent and activating reagent effect, all the other protected amino acids are extended to coupling one by one, after each extension coupling, all obtain corresponding peptide resin, the final Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 resin that obtains, 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 TFA that it is 80-95% by volume percent that described acidolysis adopts, EDT, the surplus that volume percent is 1-10% are the mixing acid hydrolysis solution acidolysis that water forms.
2. method according to claim 1, is characterized in that, protecting group is Fomc protecting group described in step 1.
3. method according to claim 1, is characterized in that, described resin carrier is Rink Amide AM resin, Rink Amide resin, Rink mbha resin or Sieber resin.
4. method according to claim 1, is characterized in that, it is 1-6:1 that the coupling of described N end has the D-alanine of protecting group and amino coupled to have the mol ratio of the aminoresin of protecting group.
5. method according to claim 1, is characterized in that, while extending coupling at every turn described in the mol ratio of amino acid and corresponding peptide resin be 1-6:1.
6. method according to claim 1, it is characterized in that, described condensation reagent is N, N-DIC, N, N-dicyclohexylcarbodiimide, phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus/organic bases, 2-(7-azepine-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-urea phosphofluoric acid ester/organic bases, benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate/organic bases, O-benzotriazole-N, N, N', the one in N'-tetramethyl-urea Tetrafluoroboric acid ester/organic bases.
7. method according to claim 6, is characterized in that, described organic bases is DIPEA, triethylamine or N-methylmorpholine.
8. method according to claim 1, is characterized in that, described activating reagent is I-hydroxybenzotriazole or N-hydroxyl-7-azepine benzotriazole.
9. method according to claim 1, is characterized in that, described esterification and the reaction solvent that extends coupling all adopt DMF.
10. method according to claim 1; it is characterized in that; described all the other protected amino acids are 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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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105085634A (en) * | 2015-08-29 | 2015-11-25 | 中肽生化有限公司 | Preparation method for degarelix |
| CN105524143A (en) * | 2016-03-10 | 2016-04-27 | 成都圣诺生物制药有限公司 | Method for synthesizing Degarelix |
| CN106749542A (en) * | 2016-12-27 | 2017-05-31 | 杭州固拓生物科技有限公司 | A kind of synthetic method of Fertirelin |
| 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 |
| CN107778354A (en) * | 2016-08-25 | 2018-03-09 | 成都圣诺生物制药有限公司 | A kind of method for synthesizing abarelix |
| CN109575109A (en) * | 2018-12-27 | 2019-04-05 | 兰州大学 | The method that fragment condensation prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 |
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| CN115141257A (en) * | 2021-03-31 | 2022-10-04 | 浙江苏泊尔制药有限公司 | Purification method of degarelix |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102329373A (en) * | 2011-09-29 | 2012-01-25 | 深圳翰宇药业股份有限公司 | Solid-phase synthetic process for degarelix |
| CN102428097A (en) * | 2009-04-24 | 2012-04-25 | 多肽实验室联合股份有限公司 | Method For The Manufacture Of Degarelix |
| CN102952174A (en) * | 2012-11-16 | 2013-03-06 | 济南环肽医药科技有限公司 | Method for synthesizing degarelix |
| CN103351428A (en) * | 2013-08-05 | 2013-10-16 | 海南双成药业股份有限公司 | Synthesis of degarelix by solid phase segment method |
| CN103992392A (en) * | 2014-05-19 | 2014-08-20 | 泰州施美康多肽药物技术有限公司 | Solid-phase synthesis method of degarelix |
-
2014
- 2014-08-27 CN CN201410427405.3A patent/CN104177478B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102428097A (en) * | 2009-04-24 | 2012-04-25 | 多肽实验室联合股份有限公司 | Method For The Manufacture Of Degarelix |
| CN102329373A (en) * | 2011-09-29 | 2012-01-25 | 深圳翰宇药业股份有限公司 | Solid-phase synthetic process for degarelix |
| CN102952174A (en) * | 2012-11-16 | 2013-03-06 | 济南环肽医药科技有限公司 | Method for synthesizing degarelix |
| CN103351428A (en) * | 2013-08-05 | 2013-10-16 | 海南双成药业股份有限公司 | Synthesis of degarelix by solid phase segment method |
| CN103992392A (en) * | 2014-05-19 | 2014-08-20 | 泰州施美康多肽药物技术有限公司 | Solid-phase synthesis method of degarelix |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105085634A (en) * | 2015-08-29 | 2015-11-25 | 中肽生化有限公司 | Preparation method for degarelix |
| CN105524143A (en) * | 2016-03-10 | 2016-04-27 | 成都圣诺生物制药有限公司 | Method for synthesizing Degarelix |
| CN105524143B (en) * | 2016-03-10 | 2019-01-22 | 成都圣诺生物制药有限公司 | A method of synthesis Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 |
| CN107778354A (en) * | 2016-08-25 | 2018-03-09 | 成都圣诺生物制药有限公司 | A kind of method for synthesizing abarelix |
| CN107778354B (en) * | 2016-08-25 | 2021-03-02 | 成都圣诺生物制药有限公司 | Method for synthesizing abarelix |
| CN106749542A (en) * | 2016-12-27 | 2017-05-31 | 杭州固拓生物科技有限公司 | A kind of synthetic method of Fertirelin |
| 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 |
| CN109575109A (en) * | 2018-12-27 | 2019-04-05 | 兰州大学 | The method that fragment condensation prepares 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 |
| CN112125956A (en) * | 2019-06-25 | 2020-12-25 | 深圳市健元医药科技有限公司 | Preparation method of degarelix |
| WO2020259714A1 (en) * | 2019-06-25 | 2020-12-30 | 深圳市健元医药科技有限公司 | Method for preparing degarelix |
| CN115141257A (en) * | 2021-03-31 | 2022-10-04 | 浙江苏泊尔制药有限公司 | Purification method of degarelix |
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