CN107778355B - Method for synthesizing cetrorelix - Google Patents
Method for synthesizing cetrorelix Download PDFInfo
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- CN107778355B CN107778355B CN201610728868.2A CN201610728868A CN107778355B CN 107778355 B CN107778355 B CN 107778355B CN 201610728868 A CN201610728868 A CN 201610728868A CN 107778355 B CN107778355 B CN 107778355B
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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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- 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 cetrorelix. The invention adopts brand new amino resin as a carrier, adopts protected D-Orn as a precursor of D-Cit, then removes side chain protecting groups to react with tert-butyl isocyanate to generate D-Cit (tBu), acidolyzes the cetrorelix peptide resin by a special trifluoroacetic acid solution containing hydrogen bromide, and simultaneously can maximally remove the side chain protecting groups tBu of the generated D-Cit (tBu), and finally the obtained cetrorelix has higher purity and total yield, avoids the generation of toxic impurities [ D-Cit (Ac) ] -cetrorelix, and has the advantages of simple and easy operation, mild conditions and higher product quality.
Description
Technical Field
The invention relates to the field of medicine synthesis, in particular to a method for synthesizing cetrorelix.
Background
Cetrorelix is a gonadotropin releasing hormone (GnRH) antagonist. GnRH binds to receptors on the cell membrane of the pituitary gland, stimulating the release of Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH). Cetrorelix competes with endogenous GnRH for receptors on pituitary cells, thereby inhibiting the release of endogenous LH and FSH. The inhibitory effect of cetrorelix was immediate after injection, with no irritation. In women, cetrorelix acts to delay the appearance of LH-surges and thus control ovulation. The effect of cetrorelix is dose dependent, with 3mg of cetrorelix maintaining 4 days of efficacy and approximately 70% inhibition at day 4. Inhibition was sustained by 0.25mg cetrorelix administered every 24 hours. Both animal and human trials have shown that endogenous LH and FSH secretion is rapidly restored after cetrorelix injection is discontinued. The clinical indication is that the patients who control the ovarian stimulation are prevented from ovulation in advance, and then the ovum collection and assisted reproductive technology treatment are carried out.
There are many reports on the preparation of cetrorelix at home and abroad, but the total yield of the synthesis processes is only about 40% at most, such as patents CN104277093A, CN104086632A, CN104610433A, CN104892732A, CN101284863A, CN101863960A, etc. In the prior art, the total yield of patent CN104086632A is the highest and reaches 45%, D-Orn (Dde) is mainly used as a precursor of D-Cit, then a protecting group Dde is removed to react with tert-butyl isocyanate to generate D-Cit (tBu), and the D-Cit (tBu) is cracked by using a traditional trifluoroacetic acid cracking solution to obtain cetrorelix. However, on the one hand, it uses toxic hydrazine hydrate to remove the D-Orn (Dde) side chain protecting group Dde; on the other hand, the totally protected cetrorelix peptide resin cannot be completely removed by adopting the traditional trifluoroacetic acid cracking solution, so that the total yield cannot be improved; the third aspect is that the appearance of toxic impurities [ D-cit (ac) ] -cetrorelix, remains incompletely avoidable.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for synthesizing cetrorelix, which has high purity and total yield, and avoids the generation of toxic impurities [ D-cit (ac) ] -cetrorelix.
In order to achieve the purpose, the invention provides the following technical scheme:
step 1, according to a cetrorelix main chain amino acid sequence, under the action of a condensation reagent and an activation reagent, protecting amino acid and amino resin to synthesize peptide resin 1, wherein D-Cit in the cetrorelix main chain amino acid sequence is replaced by protected D-Orn at a corresponding position of the peptide resin 1;
step 2, after N end of the peptide resin 1 is acetylated, removing a protected D-Orn side chain protecting group by using a protective agent, and reacting D-Orn side chain amino with tert-butyl isocyanate under the catalysis of organic base to generate D-Cit (tBu) to obtain cetrorelix peptide resin;
step 3, carrying out acidolysis on the cetrorelix peptide resin by using a trifluoroacetic acid solution containing hydrogen bromide to obtain a cetrorelix crude product;
step 4, purifying and freeze-drying the crude product of cetrorelix to obtain a pure product of cetrorelix;
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 60%. The structural formula of MBHA resin is as follows, the left round sphere representing polystyrene resin:
the cetrorelix backbone has 10 amino acids, and consists of the following components:
Ac-D-Nal1-D-Cpa2-D-Pal3-Ser4-Tyr5-D-Cit6-Leu7-Arg8-Pro9-D-Ala10-NH2
wherein, the amino group at the C end of the cetrorelix is the amino group cracked from the amino resin by adopting an acidolysis agent, and the amino group does not belong to the amino group on the amino acid.
The invention acidolyzes the cetrorelix peptide resin by a special trifluoroacetic acid solution containing hydrogen bromide, and simultaneously can remove the side chain protecting group tBu of the generated D-Cit (tBu) to the maximum, and finally the obtained cetrorelix has higher purity and total yield and has no toxic impurities [ D-Cit (Ac) ] -production of cetrorelix.
Preferably, step 1 is:
according to the sequence from the C end to the N end of the sitrorelix main chain amino acid, under the action of a condensation reagent and an activation reagent, carrying out solid phase synthesis on the following protected amino acids and amino resin according to a one-by-one coupling mode to obtain a peptide resin 1:
protected D-Ala, protected Pro, protected Arg, protected Leu, protected D-Orn, protected Ser, protected Tyr, protected D-Pal, protected D-Cpa, protected D-Nal;
wherein D-Cit in the backbone amino acid sequence of cetrorelix is replaced by a protected D-Orn at the corresponding position on peptide resin 1.
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, carboxyl and the like from reacting to generate impurities in the process of preparing a target product, for example, the side chain of Arg is protected by a Pbf protecting group, and the side chains of Tyr and Ser are protected by a tBu or Bzl protecting group; the side chain of D-Orn is protected by Boc or Fmoc 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 amino acids are as follows:
Fmoc-D-Ala, Fmoc-Pro, Fmoc-Arg (Pbf), Fmoc-Leu, protected D-Orn, Fmoc-Ser (tBu), Fmoc-Tyr (tBu), Fmoc-D-Pal, Fmoc-D-Cpa, Fmoc-D-Nal; or
Boc-D-Ala, Boc-Pro, Boc-Arg, Boc-Leu, protected D-Orn, Boc-Ser (Bzl), Boc-Tyr (Bzl), Boc-D-Pal, Boc-D-Cpa, and Boc-D-Nal.
Preferably, the protected D-Orn in the present invention is Fmoc-D-Orn (Boc) or Boc-D-Orn (Fmoc), the protected amino acid used in the present invention is less expensive than D-Orn (Dde) used in the prior art, and conventional methods for removing Boc and Fmoc protecting groups can be used for removing side chain protecting groups, and toxic reagents are not used.
Preferably, the molar ratio of each protected amino acid to the amino group in the amino resin is 1-6:1, more preferably 2.5-3.5:1, when each protected amino acid is coupled to the amino resin or the synthesized peptide resin, i.e., the ratio of the charged amount of the protected amino acid to the molar amount of the amino group 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 and the organic base in step 2 are both 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 one-by-one coupling of the amino acid is that after the first amino acid is coupled with the 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 cetrorelix 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 at each coupling is preferably 1 to 6:1, more preferably 2.5 to 3.5: 1; the coupling reaction time is preferably 60 to 300 minutes, and more preferably 120 to 180 minutes. It should be noted that the peptide resin according to the present invention refers to a peptide resin in which an arbitrary number of amino acids are linked to an amino resin in the order of cetrorelix amino acids, and includes not only the peptide resin 1 but also a plurality of peptide resins obtained during the synthesis of the peptide resin 1. The corresponding peptide resin is peptide resin 2 formed by coupling protected D-Ala and amino resin, the peptide resin 2 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 Arg, and the corresponding relations are formed by analogy, when protected Leu, protected D-Orn, protected Ser, protected Tyr, protected D-Pal, protected D-Cpa, protected D-Nal and the like are coupled with the peptide resin formed by coupling the protected amino acid on the corresponding peptide resin.
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, in the trifluoroacetic acid solution containing hydrogen bromide, the mass percent concentration of 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 acetylation according to the invention is carried out by Ac2The acetylation is accomplished by reaction of O with the deprotected N-terminal amino group of peptide resin 1.
Preferably, the purification trans-salt is specifically:
dissolving the crude product of cetrorelix in 0.1 percent TFA/water solution, filtering the solution by using a 0.45 mu m 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 a cetrorelix purified intermediate concentrated solution;
taking cetrorelix 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 a cetrorelix acetic acid water solution, and performing freeze drying to obtain a cetrorelix pure product.
The cetrorelix synthesized by the method has the advantages that the purity of a crude product is more than 85 percent, the purity of a product is more than 99.5 percent, the maximum single impurity is less than 0.15 percent, the total yield is more than 60 percent, toxic impurities [ D-Cit (Ac) ] -cetrorelix are avoided, the whole method is simple and convenient to operate, the condition is mild, and the product quality is high.
According to the technical scheme, the invention adopts brand new amino resin as a carrier, adopts protected D-Orn as a precursor of D-Cit, then removes side chain protecting groups to react with tert-butyl isocyanate to generate D-Cit (tBu), acidolytically decomposes the cetrorelix peptide resin by a special trifluoroacetic acid solution containing hydrogen bromide, and simultaneously can maximally remove the generated side chain protecting groups tBu of D-Cit (tBu), finally the obtained cetrorelix has higher purity and total yield, avoids the generation of toxic impurities [ D-Cit (Ac) ] -cetrorelix, and the whole method is simple, convenient and easy to operate, mild in condition and higher in product quality.
Detailed Description
The invention discloses a method for synthesizing cetrorelix, 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
The invention is further illustrated by the following examples.
Example 1: synthesis of D-Ala-MOBHA resin
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 an activated Fmoc-D-Ala solution, stirring at room temperature for 3 hours, pumping out the reaction solution, washing with DMF for 3 times, washing with DCM for 3 times, wherein each washing time is 3min, obtaining the Fmoc-D-Ala-MOBHA resin, deprotecting with 20% PIP/DMF solution for 25 minutes, washing and filtering to obtain D-Ala-MOBHA resin.
Example 2: synthesis of D-Ala-MOBHA resin
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 with DMF for 3 times, washing with DCM for 3 times, wherein each washing time is 3min to obtain Boc-D-Ala-MOBHA resin, deprotecting with 30% TFA/DCM solution for 30 minutes, neutralizing with DIEA/DCM solution, washing with DMF and DCM and filtering to obtain D-Ala-MOBHA resin.
Example 3: synthesis of Pro-D-Ala-MOBHA 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 the activated protected amino acid solution.
Adding the activated protected amino acid solution into the D-Ala-MOBHA resin prepared in example 1, stirring at room temperature for 3 hours, pumping out the reaction solution, washing with DMF for 3 times, washing with DCM for 3 times, washing with 3min each time, deprotecting with 20% PIP/DMF solution for 25 minutes, washing, and filtering to obtain Pro-D-Ala-MOBHA resin.
Example 4: synthesis of Pro-D-Ala-MOBHA resin
Dissolving 0.15mol of Boc-Pro 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 the activated protected amino acid solution.
The activated protected amino acid solution was added to the D-Ala-MOBHA resin obtained in example 2, stirred at room temperature for 3 hours, the reaction solution was pumped off, washed with DMF 3 times, then washed with DCM 3 times, each washing time was 3min, deprotected with 30% TFA/DCM solution for 30 minutes, neutralized with DIEA/DCM solution, washed with DMF, DCM, and filtered to give Pro-D-Ala-MOBHA resin.
Example 5: synthesis of peptide resin 1
Dissolving 0.15mol of Fmoc-Arg (Pbf) 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 the activated protected amino acid solution.
Adding the activated protected amino acid solution into the Pro-D-Ala-MOBHA resin prepared in example 3, stirring at room temperature for 3 hours, pumping out the reaction solution, washing with DMF for 3 times, washing with DCM for 3min each time, deprotecting with 20% PIP/DMF solution for 25 minutes, washing and filtering to complete the grafting of Fmoc-Arg (Pbf).
The same method is used for grafting Fmoc-Leu, Fmoc-D-Orn (Boc), Fmoc-Ser (tBu), Fmoc-Tyr (tBu), Fmoc-D-Pal, Fmoc-D-Cpa, Fmoc-D-Nal and Ac2After O (for acetylation), the mixture is washed with DMF and DCM and filtered to obtain peptide resin 1[ Ac-D-Nal-D-Cpa-D-Pal-Ser (tBu) -Tyr (tBu) -D-Orn (tBu) -Leu-Arg (Pbf) -Pro-D-A la-MOBHA resin]。
Example 6: synthesis of peptide resin 1
Dissolving 0.15mol of Boc-Arg 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 the activated protected amino acid solution.
The activated protected amino acid solution was added to the Pro-D-Ala-MOBHA resin obtained in example 4, stirred at room temperature for 3 hours, the reaction solution was aspirated, after 3 washes with DMF, DCM was washed 3 times for 3min each, then deprotected with 30% TFA/DCM solution for 30 minutes, neutralized with DIEA/DCM solution, washed with DMF, DCM and filtered to complete Boc-Arg ligation.
Boc-Leu, Boc-D-Orn (Fmoc), Boc-Ser (Bzl), Boc-Tyr (Bzl), Boc-D-Pal, Boc-D-Cpa, Boc-D-Nal and Ac were ligated in the same way2O (for acetylation), washing with DMF and DCM, and filtering to obtain peptide resin 1[ Ac-D-Nal-D-Cpa-D-Pal-Ser (Bzl) -Tyr (Bzl) -D-Orn (Fmoc) -Leu-Arg-Pro-D-Ala-MOBHA resin]。
Example 7: synthesis of cetrorelix peptide resin
The peptide resin 1 prepared in example 5 was deprotected with 30% TFA/DCM solution for 30 min, neutralized with DIEA/DCM solution, washed with DMF, DCM and filtered to recover (removal of the side chain protecting group of D-Orn (Boc)).
Dissolving 0.2mol of tert-butyl isocyanate and 0.2mol of DIEA with a proper amount of DMF, adding the solution into the peptide resin, stirring the solution at room temperature for reaction overnight, pumping out the reaction solution, washing the solution with DMF for 6 times, and washing the solution with DCM for 3 times, wherein the washing time is 3min each time, so as to obtain the cetrorelix peptide resin [ Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-Cit (tBu) -Leu-Arg-Pro-D-Ala-MOBHA resin ]
Example 8: synthesis of cetrorelix peptide resin
The peptide resin 1 obtained in example 6 was taken, deprotected with 20% PIP/DMF for 25 min, and filtered by washing with DMF, DCM, for further use (removal of the side chain protecting group of D-Orn (Fmoc)).
Dissolving 0.2mol of tert-butyl isocyanate and 0.2mol of DIEA with a proper amount of DMF, adding the mixture into the peptide resin, stirring the mixture at room temperature for reacting overnight, pumping out the reaction solution, washing the mixture for 3 times by DMF, washing the mixture for 3 times by DCM for 3min, deprotecting the mixture by using a 30% TFA/DCM solution for 30 min, neutralizing the mixture by using the DIEA/DCM solution, washing the neutralized mixture by DMF and DCM, and filtering the washed mixture to obtain the cetrorelix peptide resin [ Ac-D-Nal-D-Cpa-D-Pal-Ser (Bzl) -Tyr (Bzl) -D-cit (tBu) -Leu-Arg-Pro-D-Ala-MOB HA resin ]
Example 9: preparation of crude cetrorelix product
Taking the cetrorelix peptide resin prepared in the example 7, adding 8% HBr/TFA solution (acidolysis solution 10mL/g cetrorelix resin), stirring and reacting for 6 hours, filtering and collecting filtrate, washing the resin with a small amount of TFA for 3 times, merging the filtrate, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate with anhydrous ether for 3 times, and draining to obtain off-white powder, namely a cetrorelix crude product, wherein the purity of the crude product is 88.5%.
Example 10: preparation of crude cetrorelix product
Taking the cetrorelix peptide resin prepared in the example 8, adding 8% HBr/TFA solution (acidolysis solution 10mL/g cetrorelix resin), stirring and reacting for 6 hours, filtering and collecting filtrate, washing the resin with a small amount of TFA for 3 times, merging the filtrate, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate with anhydrous ether for 3 times, and draining to obtain off-white powder, namely crude cetrorelix, wherein the purity of the crude product is 85.6%.
Example 11: purification trans-salt of crude cetrorelix
Taking the crude product of cetrorelix obtained in the example 9, dissolving the crude product of cetrorelix by using 20 percent acetic acid solution, filtering the solution by using a 0.45 mu m microporous filter membrane, and purifying the solution 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 a cetrorelix purified intermediate concentrated solution;
taking cetrorelix 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 reversed phase C18 with a chromatographic filler of 10 mu m for purification and a chromatographic column flow rate of 77mm x 250mm is 90mL/min, performing gradient elution and a circular sample loading method, loading the sample into the chromatographic column, starting mobile phase elution, collecting a spectrum, observing the change of the absorbance, collecting a main salt exchange peak, detecting the purity by using an analytical liquid phase, combining main salt exchange peak solutions, performing reduced pressure concentration to obtain a cetrorelix acetic acid water solution, and performing freeze drying to obtain 45.3g of a cetrorelix pure product.
The total yield is 63.3% by HPLC detection, and the molecular weight is as follows: 1432.2, purity: 99.6 percent, the maximum single impurity is 0.12 percent, and toxic impurities [ D-Cit (Ac) ] -cetrorelix are not detected.
Example 12: purification trans-salt of crude cetrorelix
Dissolving the crude product of cetrorelix obtained in the example 10 by using a purification mobile phase A, filtering the solution by using a 0.45 mu m 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 a cetrorelix purified intermediate concentrated solution;
taking cetrorelix 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 reversed phase C18 with a chromatographic packing of 10 mu m for purification and a chromatographic column flow rate of 77mm x 250mm is 90mL/min, performing gradient elution and a circular sample loading method, loading the sample into the chromatographic column, starting mobile phase elution, collecting a spectrum, observing the change of the absorbance, collecting a main salt exchange peak, detecting the purity by using an analytical liquid phase, combining main salt exchange peak solutions, performing reduced pressure concentration to obtain a cetrorelix acetic acid water solution, and performing freeze drying to obtain 46.7g of a cetrorelix pure product.
The total yield is 65.2% by HPLC detection, and the molecular weight is as follows: 1432.4, purity: 99.7 percent, the maximum single impurity is 0.08 percent, and toxic impurities are not detected [ D-Cit (Ac) ] -cetrorelix.
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 cetrorelix, comprising the steps of:
step 1, according to the sequence from C end to N end of the Sitrorelix main chain amino acid, under the action of a condensation reagent and an activation reagent, carrying out solid phase synthesis on the following protected amino acid and amino resin of a formula 1 according to a one-by-one coupling mode to obtain a peptide resin 1:
protected D-Ala, protected Pro, protected Arg, protected Leu, protected D-Orn, protected Ser, protected Tyr, protected D-Pal, protected D-Cpa, protected D-Nal;
wherein D-Cit in the cetrorelix backbone amino acid sequence is replaced by a protected D-Orn at the position corresponding to peptide resin 1;
step 2, after N end of the peptide resin 1 is acetylated, removing a protected D-Orn side chain protecting group by using a protective agent, and reacting D-Orn side chain amino with tert-butyl isocyanate under the catalysis of organic base to generate D-Cit (tBu) to obtain cetrorelix peptide resin;
step 3, carrying out acidolysis on the cetrorelix peptide resin by using a trifluoroacetic acid solution containing hydrogen bromide to obtain a cetrorelix crude product; the mass percentage concentration of the hydrogen bromide in the trifluoroacetic acid solution containing the hydrogen bromide is 5-10%;
step 4, purifying, salt transferring and freeze-drying the crude product of cetrorelix to obtain a pure product of cetrorelix;
wherein, the left round ball of formula 1 represents polystyrene resin.
2. The method of claim 1, wherein the protected amino acids of step 1 are as follows:
Fmoc-D-Ala, Fmoc-Pro, Fmoc-Arg (Pbf), Fmoc-Leu, protected D-Orn, Fmoc-Ser (tBu), Fmoc-Tyr (tBu), Fmoc-D-Pal, Fmoc-D-Cpa, Fmoc-D-Nal; or
Boc-D-Ala, Boc-Pro, Boc-Arg, Boc-Leu, protected D-Orn, Boc-Ser (Bzl), Boc-Tyr (Bzl), Boc-D-Pal, Boc-D-Cpa, and Boc-D-Nal.
3. The method of any one of claims 1-2, wherein said protected D-Orn of step 1 is Fmoc-D-Orn (Boc) or Boc-D-Orn (Fmoc).
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 process according to claim 1 or 4, characterized in that 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 according to any one of claims 1-2, wherein the molar ratio of the charge of protected amino acid to amino groups of the amino resin is 1-6: 1.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999061476A1 (en) * | 1998-05-22 | 1999-12-02 | Abbott Laboratories | Peptide antiangiogenic drugs |
| CN1259959A (en) * | 1997-04-11 | 2000-07-12 | 凡林有限公司 | GnRh antagonists being modified in positions 5 and 6 |
| CN104086632A (en) * | 2014-08-05 | 2014-10-08 | 杭州诺泰制药技术有限公司 | Method for preparing cetrorelix |
| CN104610433A (en) * | 2015-02-13 | 2015-05-13 | 泰州施美康多肽药物技术有限公司 | Preparation method of cetrorelix |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1259959A (en) * | 1997-04-11 | 2000-07-12 | 凡林有限公司 | GnRh antagonists being modified in positions 5 and 6 |
| WO1999061476A1 (en) * | 1998-05-22 | 1999-12-02 | Abbott Laboratories | Peptide antiangiogenic drugs |
| CN104086632A (en) * | 2014-08-05 | 2014-10-08 | 杭州诺泰制药技术有限公司 | Method for preparing cetrorelix |
| CN104610433A (en) * | 2015-02-13 | 2015-05-13 | 泰州施美康多肽药物技术有限公司 | Preparation method of cetrorelix |
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