CN107778354B - Method for synthesizing abarelix - Google Patents
Method for synthesizing abarelix Download PDFInfo
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- CN107778354B CN107778354B CN201610728619.3A CN201610728619A CN107778354B CN 107778354 B CN107778354 B CN 107778354B CN 201610728619 A CN201610728619 A CN 201610728619A CN 107778354 B CN107778354 B CN 107778354B
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- C07—ORGANIC CHEMISTRY
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
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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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- 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 medicine synthesis, and discloses a method for synthesizing abarelix. According to the method, the side chain of N6- (1-methylethyl) lysine (Lys (ipr)) is protected by the Z protecting group, and the whole synthesis process is completed by adopting brand-new amino resin, acidolysis agent and the like, so that the obtained abarelix has high purity and total yield, low single impurity content and no toxic hydantoin degradation product, and the industrial production of the abarelix can be guided.
Description
Technical Field
The invention relates to the field of medicine synthesis, in particular to a method for synthesizing abarelix.
Background
Abarelix was developed by Praecis Pharma-ceuticals, USA, and was first marketed in the United states in 2004, month 1, and it was approved for palliative treatment of advanced symptomatic Prostate Cancer (PCA) that is not amenable to Luteinizing Hormone Releasing Hormone (LHRH) agonist treatment but to surgical resection and has one or more of the following conditions:
(1) neurological damage may occur due to tumor metastasis;
(2) blockage of the ureter or bladder outlet due to local invasion or metastatic disease;
(3) the development of severe bone pain due to tumor bone metastasis requires the dependence on narcotic analgesics;
the abarelix backbone has 10 amino acids, and consists of the following components:
Ac-D-Nal1-D-Cpa2-D-Pal3-Ser4-N-Me-Tyr5-D-Asn6-Leu7-Lys(iPr)8-Pro9-D-Ala10-NH2
abarelix reduces testosterone production in the testes by directly inhibiting Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) secretion. Because of direct inhibition of LH secretion, plasma testosterone levels do not rise first and then fall. Results of saturation binding studies showed that abarelix has a high affinity for the rat pituitary LHRH receptor.
At present, few reports about the preparation of abarelix at home and abroad are reported, and reasonable guidance cannot be provided for the industrial production of abarelix.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for synthesizing abarelix, which has high purity and total yield, and low maximum single impurity, especially avoids the generation of toxic hydantoin degradation products.
In order to achieve the purpose, the invention provides the following technical scheme:
a method of synthesizing abarelix, comprising the steps of:
step 1, carrying out coupling reaction on protected D-Ala and amino of amino resin shown in a formula 1 under the action of a condensation reagent and an activation reagent to obtain peptide resin 1;
step 2, according to the sequence from the C end to the N end of the abarelix amino acid sequence, starting from the peptide resin 1, under the action of a condensation reagent and an activation reagent, sequentially carrying out extension coupling on protected Pro, protected Lys (ipr, Z), protected Leu, protected D-Asn, protected N-Me-Tyr, protected Ser, protected D-Pal, protected D-Cpa and Ac-D-Nal one by one to obtain the abarelix peptide resin;
step 3, carrying out acidolysis on the abarelix peptide resin by an acidolysis agent to obtain an abarelix crude product;
step 4, purifying and transforming the crude Abarelix product into salt to obtain a pure Abarelix product;
wherein, the left round ball of formula 1 represents polystyrene resin. In the conventional synthesis process, the conventional Rink Amide series resin is generally adopted, the invention improves the original MBHA resin to provide a brand-new carrier resin MOBHA resin, namely the resin shown in formula 1, and the amino resin shown in formula 1 can be purchased from New science and technology materials, Inc. of Xian blue and Xiao. The invention discovers that the resin is lower than Rink Amide series resin in cost through synthesizing the product, and the total yield of the synthesized product can reach more than 55 percent. The structural formula of MBHA resin is as follows, the left round sphere representing polystyrene resin:
MBHA resin
The abarelix backbone has 10 amino acids, and consists of the following components:
Ac-D-Nal1-D-Cpa2-D-Pal3-Ser4-N-Me-Tyr5-D-Asn6-Leu7-Lys(iPr)8-Pro9-D-Ala10-NH2
wherein, the amino at the C end of the abarelix is the amino cracked from the amino resin by adopting an acidolysis agent, and the amino does not belong to the amino on the amino acid.
The invention selects Z protecting group (carbobenzoxy) to protect the side chain of N6- (1-methylethyl) lysine (Lys (iPr)), replaces Fmoc-Lys (iPr, Boc) used in the prior art, and adopts proper amino resin, acidolysis agent and the like to complete the whole synthesis process, and the obtained abarelix has higher purity and total yield, lower single impurity content and no toxic hydantoin degradation product.
The protecting group is a protecting group which needs to protect the amino acid main chain and groups interfering with synthesis, such as amino, carboxyl and the like on the side chain in the field of amino acid synthesis, and prevents the amino, the carboxyl and the like from reacting to generate impurities in the process of preparing a target product, for example, the invention protects the side chain of Asn by the Trt protecting group and protects the side chains of Tyr and Ser by the tBu protecting group; the side chain of N6- (1-methylethyl) lysine (lys (ipr)) is protected by a Z protecting group. Furthermore, in the protected amino acids involved in the process of the present invention, the N-terminus is preferably protected by Fmoc or Boc protecting group. Amino acids protected by a protecting group are collectively referred to as protected amino acids (e.g., protected D-Ala, etc.). Preferably, the protected D-Ala, protected Pro, protected Leu, protected D-Asn, protected N-Me-Tyr, protected Ser, protected D-Pal, protected D-Cpa are as follows:
Fomc-D-Ala or Boc-D-Ala; Fmoc-Pro or Boc-Pro; Fmoc-Leu; Fmoc-D-Asn (Trt); Fmoc-N-Me-Tyr (tBu); Fmoc-Ser (tBu); Fmoc-D-Pal; Fmoc-D-Cpa.
Preferably, the protected Lys (iPr, Z) of the present invention is Boc-Lys (iPr, Z), which is only one eighth of the cost of Fmoc-Lys (iPr, Boc) compared with the commonly used Fmoc-Lys (iPr, Boc), and can be more advantageous for the synthesis of abarelix in terms of overall yield and purity.
Preferably, the protected D-Ala, protected Pro, protected Lys (ipr, Z), protected Leu, protected D-Asn, protected N-Me-Tyr, protected Ser, protected D-Pal, protected D-Cpa, Ac-D-Nal, when each is coupled to an amino resin or a synthesized peptide resin, are present in a molar ratio of 1-6:1, more preferably 2.5-3.5:1, respectively, to the amino groups in the amino resin.
Preferably, the substitution value of the amino resin is 0.2 to 1.8mmol/g amino resin, more preferably 0.5 to 1.0mmol/g amino resin.
Preferably, the condensation reagent is N, N-Diisopropylcarbodiimide (DIC), N-Dicyclohexylcarbodiimide (DCC), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate/organic base (PyBOP/organic base), 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate/organic base (HATU/organic base), benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate/organic base (HBTU/organic base), O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate/organic base (TBTU/organic base). The molar usage of the condensation reagent is preferably 1 to 6 times, and more preferably 2.5 to 3.5 times of the total molar number of amino groups of the amino resin or the amino resin in the synthesized peptide resin.
It should be noted that the PyBOP/organic base, HATU/organic base, HBTU/organic base, TBTU/organic base are four two-system condensation reagents in the present invention, i.e. PyBOP, HATU, HBTU need to be combined with organic base to be one condensation reagent when in use, wherein the molar ratio of the organic base to PyBOP, HATU, HBTU, TBTU is preferably 1.3-3.0:1, more preferably 1.3-2: 1.
Preferably, the organic base in the condensation reagent is preferably N, N-Diisopropylethylamine (DIPEA), Triethylamine (TEA) or N-methylmorpholine (NMM), more preferably DIPEA.
Preferably, the activating reagent is 1-hydroxybenzotriazole (HOBt) or N-hydroxy-7-azabenzotriazole (HOAt). The amount of the activating agent is preferably 1 to 6 times, and more preferably 2.5 to 3.5 times, of the total number of moles of amino groups in the amino resin or the synthesized peptide resin.
Preferably, DMF is used as a reaction solvent for each reaction in the synthesis process.
The extension coupling refers to that after the first amino acid is coupled with amino resin, the rest amino acids are coupled with the previous coupled amino acid one by one according to the sequence from the C end to the N end of the abarelix amino acid (condensation reaction of main chain amino and carboxyl). In the coupling of the present invention, the molar ratio of the protected amino acid to the corresponding peptide resin per elongation coupling is preferably 1-6:1, more preferably 2.5-3.5: 1; the coupling reaction time is preferably 60 to 300 minutes, and more preferably 120 to 180 minutes. The peptide resin of the present invention refers to a peptide resin formed by connecting any number of amino acids to an amino resin according to the abarelix amino acid sequence, and includes not only peptide resin 1 and abarelix peptide resin. The corresponding peptide resin is peptide resin 1 formed by coupling protected D-Ala and amino resin, the peptide resin 1 is corresponding peptide resin formed by coupling protected Pro, the peptide resin coupled with protected Pro is corresponding peptide resin formed by extension coupling of protected Lys (ipr, Z), and the corresponding relations are formed by the analogy of protected Leu, protected D-Asn, protected N-Me-Tyr, protected Ser, protected D-Pal, protected D-Cpa, Ac-D-Nal and the like and peptide resin formed by coupling the protected amino acid.
In the extension coupling, since each amino acid has a protecting group at the N-terminus, it is common knowledge to those skilled in the art that the protecting group at the N-terminus needs to be removed before coupling. The invention preferably uses PIP/DMF (piperidine/N, N-dimethylformamide) mixed solution to remove the Fomc protecting group at the N end, wherein the mixed solution contains 10-30% (V) of piperidine and the balance of DMF. The time for removing the N-terminal protecting group is preferably 10 to 60 minutes, and preferably 15 to 25 minutes. The dosage of the reagent for removing the N-terminal protecting group is preferably 10mL/g of peptide resin; according to the invention, the N-terminal Boc protecting group is preferably removed by using a TFA/DCM (trifluoroacetic acid/dichloromethane) mixed solution, the trifluoroacetic acid content in the mixed solution is 20-60% (V/V), preferably 25-35% (V/V), the time for removing the N-terminal protecting group is preferably 10-50 minutes, preferably 25-35 minutes, and the dosage of the N-terminal protecting group removing reagent is preferably 10mL/g of peptide resin.
Preferably, the acid hydrolysis agent is a hydrogen bromide trifluoroacetic acid (TFA) solution, wherein: the mass percentage concentration of the hydrogen bromide is preferably 5-10 wt%, and more preferably 6-7 wt%; the dosage of the acidolysis agent is 5-15 mL of acidolysis agent/g of peptide resin, and the dosage of the acidolysis agent is preferably 7-12 mL of acidolysis agent/g of peptide resin; the acidolysis time is 1-6 hours, preferably 3-4 hours.
Preferably, the purification trans-salt is specifically:
dissolving the crude Abarelix product in 0.1% TFA/water solution, filtering the solution with a 0.45-micrometer microporous filter membrane, and purifying for later use;
purifying by high performance liquid chromatography, wherein a chromatographic filler is 10 mu m reverse phase C18, a mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, a chromatographic column with the flow rate of 77mm x 250mm is 90mL/min, eluting by a gradient system, circularly sampling and purifying, sampling a crude product solution in the chromatographic column, starting the mobile phase for elution, collecting a main peak, and evaporating acetonitrile to obtain an abarelix purified intermediate concentrated solution;
taking the Abarelix purified intermediate concentrated solution, and filtering with a 0.45-micrometer filter membrane for later use;
performing salt exchange by adopting a high performance liquid chromatography, wherein a mobile phase system is 1% acetic acid/water solution-acetonitrile, a chromatographic filler for purification is reversed phase C18 with the diameter of 10 mu m, the flow rate of a chromatographic column with the diameter of 77mm × 250mm is 90mL/min, a gradient elution method and a circular sample loading method are adopted, loading the chromatographic column, starting the mobile phase elution, collecting a spectrum, observing the change of the absorbance, collecting a main salt exchange peak, detecting the purity by using an analysis liquid phase, combining main salt exchange peak solutions, concentrating under reduced pressure to obtain an abarelix acetic acid water solution, and performing freeze drying to obtain an abarelix pure product.
The abarelix synthesized by the method has the purity of over 99.5 percent, the maximum single impurity of not more than 0.13 percent and the total yield of more than 55 percent through HPLC detection, avoids the generation of toxic hydantoin degradation products, and has the advantages of simple and convenient operation of the whole method and higher product quality.
According to the technical scheme, the side chain of N6- (1-methylethyl) lysine (Lys (ipr)) is protected by the Z protecting group, and the whole synthesis process is completed by adopting brand-new amino resin, acidolysis agent and the like, so that the obtained abarelix has high purity and total yield, low single impurity content and no toxic hydantoin degradation product, and the industrial production of the abarelix can be guided.
Detailed Description
The invention discloses a method for synthesizing abarelix, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as appropriate variations and combinations of the methods described herein, may be made and the techniques of the present invention employed without departing from the spirit and scope of the invention.
In a specific embodiment of the present invention, the protected amino acids of the present invention are obtained from Yoghui bioscience, Inc., and the resins are obtained from Papaver rhoeas, Inc., and the English abbreviations used in the application documents have the Chinese meanings shown in Table 1.
TABLE 1 English abbreviation definitions
| English abbreviation | Name of Chinese | English abbreviation | Name of Chinese |
| Fmoc | 9-fluorenylmethoxycarbonyl group | Asn | D-asparagine |
| tBu | Tert-butyl radical | D-Cpa | 4-chloro-D-phenylalanine |
| Boc | Boc-acyl | D-Ala | D-alanine |
| Z | Benzyloxycarbonyl group | D-Aph | 4-amino-D-phenylalanine |
| Trt | Trityl radical | Lys(iPr) | N6- (1-methylethyl) lysine |
| Ser | Serine | D-Nal | 3- (2-naphthyl) -D-alanine |
| Leu | Leucine | D-Pal | 3- (3-pyridinyl) -D-alanine |
| Ac | Acetyl group | Tyr | Tyrosine |
The invention is further illustrated by the following examples.
Example 1: synthesis of peptide resin 1
Dissolving 0.15mol of Fmoc-D-Ala and 0.15mol of HOBt by using a proper amount of DMF; and adding 0.15mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.
Taking 0.05mol of MOBHA resin (the substitution value is about 0.6mmol/g), swelling with DMF for 25 minutes, washing and filtering, adding the activated solution, stirring at room temperature for reaction for 3 hours, pumping out the reaction solution, washing with DMF for 3 times, washing with DCM for 3 times, wherein the washing time is 3min each time, obtaining Fmoc-D-Ala-MOBHA resin, namely the peptide resin 1, removing Fmoc protection with 20% PIP/DMF solution for 25 minutes before carrying out the next coupling reaction, washing and filtering to obtain the D-Ala-MOBHA resin.
Example 2: synthesis of peptide resin 1
Dissolving 0.15mol of Boc-D-Ala and 0.15mol of HOBt with a proper amount of DMF; and adding 0.15mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.
Taking 0.05mol of MOBHA resin (the substitution value is about 0.6mmol/g), swelling with DMF for 25 minutes, washing and filtering, adding an activated Fmoc-D-Ala solution, stirring at room temperature for 3 hours, pumping out the reaction solution, washing 3 times with DMF, washing 3 times with DCM, wherein each washing time is 3min, obtaining Boc-D-Ala-MOBHA resin, namely peptide resin 1, deprotecting with 30% TFA/DCM solution for 30 minutes, neutralizing with DIEA/DCM solution, washing and filtering with DMF and DCM, and obtaining D-Ala-MOBHA resin.
Example 3: synthesis of Abarelix peptide resin
Dissolving 0.15mol of Fmoc-Pro and 0.15mol of HOBt in a proper amount of DMF; and adding 0.15mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.
Adding the activated Fmoc-Pro solution into the peptide resin 1 obtained in example 1, stirring at room temperature for reaction for 3 hours, pumping out the reaction solution, washing with DMF for 3 times, washing with DCM for 3 minutes each time, removing Fmoc protection with 20% PIP/DMF solution for 25 minutes, washing and filtering to obtain Pro-D-Ala-MOBHA resin.
Boc-Lys (iPr, Z), Fmoc-Leu, Fmoc-D-Asn (Trt), Fmoc-N-Me-Tyr (tBu), Fmoc-Ser (tBu), Fmoc-D-Pal, Fmoc-D-Cpa and Ac-D-Nal are sequentially added in the same method, and the Abarelix peptide resin, Ac-D-Nal-D-Cpa-D-Pal-Ser (tBu) -N-Me-Tyr (tBu) -D-Asn (Trt) -Leu-Lys (iPr, Z) -Pro-D-Ala-MOBHA resin are obtained by washing and filtering.
Example 4: synthesis of Abarelix peptide resin
Dissolving 0.15mol of Boc-Pro and 0.15mol of HOBt by using a proper amount of DMF; and adding 0.15mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.
Adding the activated Boc-Pro solution into the peptide resin 1 obtained in example 1, stirring at room temperature for reaction for 3 hours, pumping out the reaction solution, washing with DMF for 3 times, washing with DCM for 3min each time, deprotecting with 30% TFA/DCM solution for 30 minutes, neutralizing with DIEA/DCM solution, washing with DMF and DCM, and filtering to obtain Pro-D-Ala-MBHA resin.
Boc-Lys (iPr, Z), Fmoc-Leu, Fmoc-D-Asn (Trt), Fmoc-N-Me-Tyr (tBu), Fmoc-Ser (tBu), Fmoc-D-Pal, Fmoc-D-Cpa and Ac-D-Nal are sequentially added in the same method, and the Abarelix peptide resin, Ac-D-Nal-D-Cpa-D-Pal-Ser (tBu) -N-Me-Tyr (tBu) -D-Asn (Trt) -Leu-Lys (iPr, Z) -Pro-D-Ala-MOBHA resin are obtained by washing and filtering.
Example 5: preparation of crude Abarelix
Taking the abarelix peptide resin prepared in the example 3, adding 8% HBr/TFA solution (acidolysis solution 10mL/g abarelix resin), stirring and reacting for 6 hours, filtering and collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate with anhydrous ether for 3 times, and draining to obtain white-like powder, namely a crude product of abarelix, wherein the purity of the crude product is 79.3%.
Example 6: preparation of crude Abarelix
Taking the abarelix peptide resin prepared in the example 4, adding 8% HBr/TFA solution (acidolysis solution 10mL/g abarelix resin), stirring and reacting for 6 hours, filtering and collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate with anhydrous ether for 3 times, and draining to obtain white-like powder, namely a crude product of abarelix, wherein the purity of the crude product is 77.4%.
Example 7: purification and trans-salt conversion of crude Abarelix
Taking the crude Abarelix product obtained in the example 5, dissolving the Abarelix product in 20 percent acetic acid solution, filtering the solution by using a 0.45 mu m microporous membrane, and purifying for later use;
purifying by high performance liquid chromatography, wherein a chromatographic filler is 10 mu m reverse phase C18, a mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, a chromatographic column with the flow rate of 77mm x 250mm is 90mL/min, eluting by a gradient system, circularly sampling and purifying, sampling a crude product solution in the chromatographic column, starting the mobile phase for elution, collecting a main peak, and evaporating acetonitrile to obtain an abarelix purified intermediate concentrated solution;
taking the Abarelix purified intermediate concentrated solution, and filtering with a 0.45-micrometer filter membrane for later use;
performing salt exchange by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the purification is performed by reversed phase C18 with chromatographic packing of 10 μm, the flow rate of a chromatographic column of 77mm × 250mm is 90mL/min, gradient elution and circular sample loading method are adopted, the sample is loaded in the chromatographic column, the mobile phase elution is started, the chromatogram is collected, the change of the absorbance is observed, the main peak of salt exchange is collected and the purity is detected by analyzing the liquid phase, the main peak solutions of salt exchange are combined, the concentration is performed under reduced pressure to obtain the aqueous solution of abarelix acetic acid, and freeze drying is performed to obtain 39.4g abarelix pure product
The total yield was 55.6%, molecular weight: 1417.2, purity: 99.6%, maximum single impurity of 0.13%, no toxic hydantoin degradation products were detected.
Example 8: purification and trans-salt conversion of crude Abarelix
Taking the crude Abarelix product obtained in the example 6, dissolving the Abarelix product by using a purification mobile phase A, and filtering the solution by using a 0.45 mu m microporous filter membrane to purify the Abarelix product for later use;
purifying by high performance liquid chromatography, wherein a chromatographic filler is 10 mu m reverse phase C18, a mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, a chromatographic column with the flow rate of 77mm x 250mm is 90mL/min, eluting by a gradient system, circularly sampling and purifying, sampling a crude product solution in the chromatographic column, starting the mobile phase for elution, collecting a main peak, and evaporating acetonitrile to obtain an abarelix purified intermediate concentrated solution;
taking the Abarelix purified intermediate concentrated solution, and filtering with a 0.45-micrometer filter membrane for later use;
performing salt exchange by adopting a high performance liquid chromatography, wherein a mobile phase system is 1% acetic acid/water solution-acetonitrile, a chromatographic filler for purification is reversed phase C18 with the diameter of 10 mu m, the flow rate of a chromatographic column with the diameter of 77mm × 250mm is 90mL/min, a gradient elution method and a circular sample loading method are adopted, loading the chromatographic column, starting the mobile phase elution, collecting a spectrum, observing the change of the absorbance, collecting a main salt exchange peak, detecting the purity by using an analysis liquid phase, combining main salt exchange peak solutions, concentrating under reduced pressure to obtain an abarelix acetic acid water solution, and performing freeze drying to obtain 41.7g of an abarelix pure product.
The total yield is 58.9%, molecular weight: 1417.0, purity: 99.5%, maximum single impurity 0.09%, no toxic hydantoin degradation products were detected.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A method of synthesizing abarelix, comprising the steps of:
step 1, carrying out coupling reaction on protected D-Ala and amino of amino resin shown in a formula 1 under the action of a condensation reagent and an activation reagent to obtain peptide resin 1;
step 2, according to the sequence from the C end to the N end of the abarelix amino acid sequence, starting from the peptide resin 1, under the action of a condensation reagent and an activation reagent, sequentially carrying out extension coupling on protected Pro, protected Lys (ipr, Z), protected Leu, protected D-Asn, protected N-Me-Tyr, protected Ser, protected D-Pal, protected D-Cpa and Ac-D-Nal one by one to obtain the abarelix peptide resin;
step 3, carrying out acidolysis on the abarelix peptide resin by using an acidolysis agent to obtain an abarelix crude product, wherein the acidolysis agent is a trifluoroacetic acid solution of hydrogen bromide, and the mass percentage concentration of the hydrogen bromide in the acidolysis agent is 5-10%;
step 4, purifying and transforming the crude Abarelix product into salt to obtain a pure Abarelix product;
wherein, the left round ball of formula 1 represents polystyrene resin.
2. The method of claim 1, wherein the protected D-Ala, protected Pro, protected Leu, protected D-Asn, protected N-Me-Tyr, protected Ser, protected D-Pal, protected D-Cpa are as follows:
Fomc-D-Ala or Boc-D-Ala; Fmoc-Pro or Boc-Pro; Fmoc-Leu; Fmoc-D-Asn (Trt); Fmoc-N-Me-Tyr (tBu); Fmoc-Ser (tBu); Fmoc-D-Pal; Fmoc-D-Cpa.
3. The method of claim 1, wherein said protected Lys (ipr, Z) is Boc-Lys (ipr, Z).
4. The method of claim 1, wherein the condensation reagent is one of N, N-diisopropylcarbodiimide, N, N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate/organic base, 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate/organic base, benzotriazol-N, N, N ', N' -tetramethyluronium hexafluorophosphate/organic base, O-benzotriazol-N, N, N ', N' -tetramethyluronium tetrafluoroborate/organic base.
5. The method of claim 4, wherein the organic base is N, N-diisopropylethylamine, triethylamine, or N-methylmorpholine.
6. The method of claim 1, wherein the activating reagent is 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole.
7. The method of claim 1, wherein the molar ratio of protected D-Ala, protected Pro, protected Lys (ipr, Z), protected Leu, protected D-Asn, protected N-Me-Tyr, protected Ser, protected D-Pal, protected D-Cpa, and protected Ac-D-Nal to amino group of the amino resin is 1-6:1, respectively.
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| US5843902A (en) * | 1995-12-15 | 1998-12-01 | Praecis Pharmaceuticals Incorporated | Methods for treating prostate cancer with LHRH antagonists |
| WO2005117934A1 (en) * | 2004-05-31 | 2005-12-15 | Smart Drug Systems Inc | Sustained release composition |
| CN104177478A (en) * | 2014-08-27 | 2014-12-03 | 成都圣诺生物科技股份有限公司 | Method for synthesizing 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 |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5843902A (en) * | 1995-12-15 | 1998-12-01 | Praecis Pharmaceuticals Incorporated | Methods for treating prostate cancer with LHRH antagonists |
| US6153586A (en) * | 1995-12-15 | 2000-11-28 | Praecis Pharmaceuticals, Inc. | Methods for treating prostate cancer with LHRH-R antagonists |
| WO2005117934A1 (en) * | 2004-05-31 | 2005-12-15 | Smart Drug Systems Inc | Sustained release composition |
| CN104177478A (en) * | 2014-08-27 | 2014-12-03 | 成都圣诺生物科技股份有限公司 | Method for synthesizing 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 |
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