A preparation method of ganirelix acetate

Abstract

The invention discloses a method for preparing ganirelix acetate, comprising the following steps: 1) coupling an amino resin with an amino acid to obtain a linear peptide resin; 2) subjecting the linear peptide resin obtained in 1) to an acetylation reaction; 3) cracking the linear peptide resin obtained after the acetylation reaction in 2) to obtain a linear peptide; 4) modifying the linear peptide in 3) with ethylaminoethylimino-1H-pyrazole under alkaline conditions to obtain a crude ganirelix acetate peptide; 5) purifying the crude ganirelix acetate peptide. The invention avoids using Fmoc-HArg(Et)2-OH and Fmoc-D-HArg(Et)2-OH, which are high in synthesis cost and easy to decompose, as raw materials, avoids the need to continuously add alkali during the reaction process to adjust the pH to 7.5-10.5 for reaction, has a long reaction time, is cumbersome to operate, and has the disadvantages of low purity of the crude peptide.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide drug production and relates to a method for preparing ganirelix acetate. Background Art

[0002] Ganirelix acetate is an antagonist of gonadotropin-releasing hormone (GnRH), which inhibits pituitary secretion of LH more significantly than FSH, thereby reducing the production of sex hormones. For women with ovarian hyperstimulation, this product can prevent LH fluctuations and related stimulation, and increase implantation and pregnancy rates. Therefore, Ganirelix acetate has high medicinal value and broad market prospects. Ganirelix acetate injection was developed and produced by Merck, and was launched in the United States in 1999. It was approved by the China Food and Drug Administration for marketing in China in 2013 under the trade name ORGALUTRAN. The structure of Ganirelix contains 10 amino acids, and the peptide sequence is as follows:

[0003] Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-(diet)Har-Leu-(diet)Har-Pro-D-Ala-NH2

[0004] At present, the preparation method of Ganirelix has been reported both at home and abroad: for example, international patents US 4801577, US 5212288, and US 5767082 disclose a solid phase synthesis method of the Boc strategy, but such a method has backward technology, low yield, and large environmental pollution, and is not suitable for the industrial production of Ganirelix acetate. The solid phase synthesis method adopted by domestic patents CN102584945A and CN110563812A requires the use of Fmoc-HArg (Et) 2-OH and Fmoc-D-HArg (Et) 2-OH as raw materials. These two amino acids have high synthesis costs and are easy to decompose. In the process of synthesizing Ganirelix peptide resin, multiple coupling reactions are required to complete the reaction, which is time-consuming and leads to the generation of more impurities, which is not conducive to industrialized scale-up production. Patent CN104844694A uses Fmoc-Lys(Boc)-OH and Fmoc-D-Lys(Boc)-OH to replace Fmoc-HArg(Et)2-OH and Fmoc-D-HArg(Et)2-OH respectively to synthesize a crude peptide precursor, which is then reacted with the reagent ethylaminoethyliminomethanesulfonic acid to obtain ganirelix. In the reaction of the modified linear peptide, it is necessary to continuously add alkali to adjust the pH to 7.5-10.5 during the reaction, and the time used for the modified linear peptide reaction is 8.0-48.0h. The operation is cumbersome, the reaction time is long, and the purity of the crude peptide is not high.

[0005] In summary, in view of the above problems existing in the prior art, it is necessary to invent a new method for preparing ganirelix acetate. Summary of the invention

[0006] In order to solve the problems raised in the above background technology, the purpose of the present invention is to provide a method for preparing ganirelix acetate. In the preparation method of the present invention, the raw material price is low, the coupling efficiency is high, the reaction operation is simple, and the obtained crude peptide has high purity.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a method for preparing ganirelix acetate, comprising the following steps:

[0008] 1) amino resin is coupled with amino acid to obtain linear peptide resin;

[0009] 2) subjecting the linear peptide resin obtained in 1) to acetylation reaction;

[0010] 3) cleaving the linear peptide resin obtained after the acetylation reaction in 2) to obtain a linear peptide;

[0011] 4) The linear peptide in 3) is modified with ethylaminoethylimino-1H-pyrazole under alkaline conditions to obtain crude ganirelix acetate peptide.

[0012] Further, the amino resin in 1) includes Rink Amide resin, Rink Amide AM resin or Rink Amide MBHA resin; preferably, the amino resin in 1) is Rink Amide resin;

[0013] The substitution degree of the amino resin in 1) is 0.4-1.2 mmol / g; preferably, the substitution degree of the amino resin in 1) is 0.6-1.0 mmol / g.

[0014] Furthermore, the preparation method of the linear peptide resin described in 1) is:

[0015] a. Add the amino resin to the solid phase reaction column, swell it with a solvent, remove the Fmoc protecting group and then wash it;

[0016] b. Dissolve Fmoc-D-Ala-OH and a coupling agent in a solvent and activate them, then add them together to a solid phase reaction column, carry out coupling reaction at 20°C-30°C for 1-3 hours, and wash after the reaction; then remove the Fmoc protecting group and wash; repeat the above process, and sequentially couple amino acids Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-Pal-OH, Fmoc-D-Cpa-OH, and Fmoc-D-Nal-OH from C-terminus to N-terminus;

[0017] Preferably, the coupling reaction temperature is 25°C;

[0018] Preferably, the coupling reaction time is 2 hours;

[0019] Preferably, the solvent used for the swelling is one of N,N-dimethylformamide, N-methyl-2-pyrrolidone, and dichloromethane. More preferably, the solvent used for the swelling is N,N-dimethylformamide.

[0020] Preferably, the solvent used for washing is one of N,N-dimethylformamide, N-methyl-2-pyrrolidone and dichloromethane. More preferably, the solvent used for washing is N,N-dimethylformamide.

[0021] Preferably, the solvent used in the coupling reaction is one of N,N-dimethylformamide, N-methyl-2-pyrrolidone and dichloromethane. More preferably, the solvent used in the coupling reaction is N,N-dimethylformamide.

[0022] Preferably, the molar ratio of Fmoc-AA-OH to the synthesis scale is 3-5:1, preferably 4:1; the synthesis scale is the mass of the amino resin multiplied by the degree of substitution of the amino resin, wherein Fmoc-AA-OH is Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-Pal-OH, Fmoc-D-Cpa-OH or Fmoc-D-Nal-OH.

[0023] Furthermore, the reagent used for removing the Fmoc protecting group is a 20% piperidine / N,N-dimethylformamide solution.

[0024] Further, the coupling agent is a composition of DIPCDI and compound A or a composition of DIPEA, compound A and compound B, wherein compound A is selected from HOAt or HOBt, and compound B is selected from PyAOP, PyBOP, HATU, HBTU or TBTU. Preferably, the coupling agent is a composition of DIPCDI and compound A;

[0025] Preferably, the ratio of Fmoc-AA-OH to each component in the coupling agent is Fmoc-AA-OH:DIPCDI:A=1:1.2:1.1 or Fmoc-AA-OH:DIPEA:A:B=1:2.0:1.1:1.0 in terms of molar ratio, wherein Fmoc-AA-OH is Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-Pal-OH, Fmoc-D-Cpa-OH or Fmoc-D-Nal-OH.

[0026] Furthermore, the acetylation reaction of the linear peptide resin obtained in 1) in 2) is specifically as follows: after the amino acid coupling is completed, the Fmoc protecting group is removed and then washed, and then a mixed solution of acetic anhydride, pyridine and a solvent is added, and the reaction is carried out at 20° C.-30° C. for 1-3 hours, and the solvent is one of DMF, NMP and dichloromethane;

[0027] Preferably, the reaction time is 2 hours;

[0028] Preferably, the reaction temperature is 25°C;

[0029] Preferably, the solvent is DMF;

[0030] Preferably, in the acetylation reaction of the linear peptide resin obtained in 1) described in 2), the ratio of acetic anhydride, pyridine, solvent and synthesis scale is 15-30:15-30:15-30:15-30:1 in molar ratio, preferably 20:20:20:1; the synthesis scale is the mass of the amino resin multiplied by the degree of substitution of the amino resin.

[0031] Furthermore, the cleavage solution used in the cleavage in 3) is a mixture of TFA and H2O, the volume ratio of TFA and H2O is 95:5, and the volume weight ratio of the cleavage solution to the linear peptide resin obtained after the acetylation reaction in 2) is 6-8mL:1g; the cleavage temperature is room temperature, and the time is 2-3 hours, preferably 2.5 hours.

[0032] Further, the method described in 4) of modifying the linear peptide in 3) with ethylaminoethylimino-1H-pyrazole under alkaline conditions to obtain crude ganirelix acetate peptide is specifically: adding the linear peptide in 3) and ethylaminoethylimino-1H-pyrazole into water to dissolve, and then adjusting the pH to 8.5-10.5 for reaction, the reaction time is 4-6 hours, and the reaction temperature is 20°C-30°C;

[0033] Preferably, the reaction time is 5 hours;

[0034] Preferably, the reaction temperature is 25°C;

[0035] Preferably, the solution used for pH adjustment is a sodium hydroxide aqueous solution, and the mass fraction of the sodium hydroxide aqueous solution is 5% to 15%; preferably, the mass fraction of the sodium hydroxide aqueous solution is 10%;

[0036] Preferably, the molar ratio of the ethylaminoethylimino-1H-pyrazole to the linear peptide in 3) is 5-15:1, preferably 10:1.

[0037] Furthermore, 4) further includes the steps of purification and salt conversion of the crude ganirelix peptide, and preferably, the purification is HPLC purification.

[0038] Furthermore, the HPLC purification is divided into two steps, including the following steps: (1) the first step of purification: using 10 μm C18 as the stationary phase, the mobile phase A1 phase is a trifluoroacetic acid solution with a volume percentage of 1‰, the B phase is pure acetonitrile, and the gradient elution is 95% A1+5% B changing to 45% A1+55% B;

[0039] (2) Second step purification: 10 μm C18 was used as the stationary phase, the mobile phase A2 was 50 mmol / L sodium perchlorate monohydrate solution, the pH was adjusted to 2.50±0.05 with phosphoric acid, the mobile phase B was pure acetonitrile, and the gradient elution was changed from 95% A2+5% B to 50% A2+50% B;

[0040] The salt transfer method comprises the following steps: using 10 μm C18 as the stationary phase, the mobile phase A3 phase is an acetic acid solution with a volume percentage of 0.2%, the pH value is adjusted to 6.2-6.5 with ammonia water, the A4 phase is an acetic acid solution with a volume percentage of 0.2%, the B phase is pure acetonitrile, after 95% A3+5% B isocratic elution, the mobile phase type and gradient are changed to 95% A4+5% B for gradient elution, and the gradient elution is changed from 95% A4+5% B to 50% A4+50% B;

[0041] Preferably, the gradient elution in the first step of purification is specifically as follows: the sample is loaded after equilibration for 5 minutes using a 97% A1 + 3% B mobile phase, the gradient changes to 95% A1 + 5% B after loading, the gradient elution changes from 95% A1 + 5% B to 82% A1 + 18% B within 2 minutes, and then the gradient elution changes from 82% A1 + 18% B to 68% A1 + 32% B within 60 minutes, after the above process completes the elution of the sample, the mobile phase gradient changes from 68% A1 + 32% B to 55% A1 + 45% B for 6 minutes, and then the mobile phase gradient changes from 55% A1 + 45% B to 45% A1 + 55% B for 6 minutes, and finally after 6 minutes the mobile phase gradient returns to the initial 97% A1 + 3% B, and this process completes the flushing of the chromatographic column;

[0042] The gradient elution in the second step of purification is specifically as follows: the sample is loaded after equilibration for 5 minutes using a 95% A2 + 5% B mobile phase, and the gradient elution changes from 95% A2 + 5% B to 75% A2 + 25% B within 4 minutes after loading the sample, and then the gradient elution changes from 75% A2 + 25% B to 63% A2 + 37% B within 60 minutes. After the above process completes the elution of the sample, the mobile phase gradient changes from 63% A2 + 37% B to 50% A2 + 50% B within 1 minute, and then the mobile phase gradient remains unchanged at 50% A2 + 50% B within 4 minutes, and finally after 1 minute, the mobile phase gradient returns to the initial 95% A2 + 5% B, and this process completes the flushing of the chromatographic column;

[0043] The elution in the salt transfer method is specifically as follows: the sample is loaded after balancing with a mobile phase of 95% A3+5% B for 5 minutes, and then the mobile phase gradient is 95% A3+5% B for isocratic elution for 20 minutes, and then the mobile phase type and gradient are changed to 95% A4+5% B for gradient elution, specifically, the gradient elution is changed from 95% A4+5% B to 75% A4+25% B for 10 minutes, and then the gradient elution is changed from 75% A3+25% B to 63% A4+37% B for 30 minutes. After the above process completes the elution and salt transfer of the sample, the mobile phase gradient is changed from 63% A4+37% B to 50% A4+50% B for 5 minutes, and then the mobile phase gradient is kept unchanged at 50% A4+50% B within 5 minutes, and finally the mobile phase gradient returns to the initial 95% A3+5% B after 5 minutes, and this process completes the flushing of the chromatographic column.

[0044] The beneficial effects of the present invention are as follows: the present invention adopts an amino resin as a solid phase carrier to couple with an amino acid to obtain a peptide resin, and then acetylates and cleaves to obtain a linear peptide, and then reacts with ethylaminoethylimino-1H-pyrazole under alkaline conditions to obtain a method for obtaining ganirelix acetate, thereby avoiding the use of Fmoc-HArg(Et)2-OH and Fmoc-D-HArg(Et)2-OH, which have high synthesis costs and are easily decomposed, as raw materials. Using ethylaminoethylimino-1H-pyrazole as a raw material instead of the reagent ethylaminoethyliminomethanesulfonic acid to obtain ganirelix acetate avoids the need to continuously add alkali during the reaction process to adjust the pH to 7.5-10.5 for reaction, which has the disadvantages of long reaction time, complicated operation and low purity of the obtained crude peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is a flow chart of the preparation of ganirelix acetate in an embodiment of the present invention;

[0046] Figure 2 This is the HPLC spectrum of the linear peptide of ganirelix acetate in Example 3 of the present invention;

[0047] Figure 3 This is the HPLC spectrum of Ganirelix acetate peptide in Example 5 of the present invention. DETAILED DESCRIPTION

[0048] In order to better understand the content of the present invention, the content of the present invention is further described below in conjunction with specific implementation methods, but the protection content of the present invention is not limited to the following embodiments.

[0049] The meanings of the abbreviations used in the specification and claims are listed in the following table:

[0050]

[0051]

[0052] The preparation flow chart of Ganirelix acetate is as follows Figure 1 shown.

[0053] Example 1: Coupling of amino resin with amino acid to obtain linear peptide resin

[0054] 53.5 g of Rink Amide resin with a substitution degree of 0.6 mmol / g was loaded into the solid phase reaction column, the amino resin was swollen with DMF for half an hour, DBLK was added for deprotection for 5 min + 7 min, the resin was washed with DMF 6 times, and the result of the resin detection with ninhydrin showed positive.

[0055] Weigh Fmoc-D-Ala-OH (30.0 g, 96.4 mmol) and HOBt (14.3 g, 106.04 mmol) and dissolve them in DMF. Add DIC (17.9 mL, 115.6 mmol) to activate for 5 min, then add the mixture to the reaction column and react at room temperature for 2 hours. The ninhydrin test is negative. Wash the resin 3 times with DMF, add DBLK for deprotection for 5 min + 7 min, wash the resin 6 times with DMF, and the ninhydrin test result is positive.

[0056] Repeat the above steps to complete the coupling of Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-Pal-OH, Fmoc-D-Cpa-OH, and Fmoc-D-Nal-OH in sequence by coupling one by one.

[0057] Example 2: Peptide resin acetylation

[0058] After the amino acid coupling was completed, DBLK was added for deprotection for 5min+7min, and the resin was washed with DMF 6 times. The ninhydrin test was positive. 60.2ml of acetic anhydride and 51.7ml of pyridine were weighed and diluted to 150ml with DMF and added to the solid phase reaction column for reaction at room temperature for 2 hours. The reaction was completed and the ninhydrin test was negative. The resin was washed with DMF 6 times, shrunk with methanol 3 times, and vacuum-dried to obtain 124.3g of peptide resin.

[0059] Example 3: Cleavage to obtain linear peptides

[0060] Add the above peptide resin to a 2L flask, add the cleavage reagent (TFA:H2O (volume ratio) = 95:5, 992mL) pre-frozen to -15°C, stir at room temperature for 2.5 hours, filter the resin, and collect the filtrate. Wash the resin with a small amount of TFA and combine the filtrate. Slowly add the filtrate to 10L of ice ether for precipitation. Centrifuge, wash with ether twice, and blow dry with nitrogen to obtain 62.86g of linear peptide, with a linear peptide purity of 93.70% and a yield of 97.2%.

[0061] After testing, the MS data of Ganirelix acetate linear peptide are: 1373.561 (M+1); 1395.632 (M+1), and the HPLC spectrum is as follows Figure 2 As shown, the target peak of the product is at the retention value T=17.738, and the purity is 93.70%.

[0062] Example 4: Modification of linear peptide to obtain crude ganirelix peptide

[0063] 4.1 Add the above crude peptide (3.1 g, 2 mmol) and ethylaminoethylimino-1H-pyrazole (1.99 g, 12 mmol) into a 500 ml flask and dissolve them in 50 ml purified water. Adjust the pH of the reaction solution to 8.5 with 10% sodium hydroxide aqueous solution and react at room temperature for 4 hours. The reaction process is monitored by HPLC, and the purity of the product in the reaction solution is 72%.

[0064] 4.2 Add the above crude peptide (3.1 g, 2 mmol) and ethylaminoethylimino-1H-pyrazole (1.99 g, 12 mmol) into a 500 ml flask and dissolve them in 50 ml purified water. Adjust the pH of the reaction solution to 8.5 with 10% sodium hydroxide aqueous solution and react at room temperature for 5 hours. The reaction process is monitored by HPLC and the purity of the product in the reaction solution is 80%.

[0065] 4.3 Add the above crude peptide (3.1 g, 2 mmol) and ethylaminoethylimino-1H-pyrazole (1.99 g, 12 mmol) into a 500 ml flask and dissolve them in 50 ml purified water. Adjust the pH of the reaction solution to 8.5 with 10% sodium hydroxide aqueous solution and react at room temperature for 6 hours. The reaction process is monitored by HPLC, and the purity of the product in the reaction solution is 78%.

[0066] 4.4 Add the above crude peptide (3.1 g, 2 mmol) and ethylaminoethylimino-1H-pyrazole (1.99 g, 12 mmol) into a 500 ml flask and dissolve them in 50 ml purified water. Adjust the pH of the reaction solution to 9.0 with 10% sodium hydroxide aqueous solution and react at room temperature for 5 hours. The reaction process was monitored by HPLC and the purity of the product in the reaction solution was 81%.

[0067] 4.5 The crude peptide (3.1 g, 2 mmol) and ethylaminoethylimino-1H-pyrazole (1.99 g, 12 mmol) were added to a 500 ml flask and dissolved with 50 ml purified water. The pH of the reaction solution was adjusted to 9.5 with 10% sodium hydroxide aqueous solution and the reaction was carried out at room temperature for 5 hours. The reaction process was monitored by HPLC and the purity of the product in the reaction solution was 85%.

[0068] 4.6 The crude peptide (3.1 g, 2 mmol) and ethylaminoethylimino-1H-pyrazole (1.99 g, 12 mmol) were added to a 500 ml flask and dissolved with 50 ml purified water. The pH of the reaction solution was adjusted to 10.0 with 10% sodium hydroxide aqueous solution and the reaction was carried out at room temperature for 5 hours. The reaction process was monitored by HPLC and the purity of the product in the reaction solution was 76%.

[0069] 4.7 The crude peptide (3.1 g, 2 mmol) and ethylaminoethylimino-1H-pyrazole (1.99 g, 12 mmol) were added to a 500 ml flask and dissolved with 50 ml purified water. The pH of the reaction solution was adjusted to 10.5 with 10% sodium hydroxide aqueous solution and the reaction was carried out at room temperature for 5 hours. The reaction process was monitored by HPLC and the purity of the product in the reaction solution was 74%.

[0070] Through the above series of control experiments, it can be concluded that the optimal reaction conditions are to adjust the pH of the reaction solution to 9.5 with a 10% NaOH aqueous solution and to react for 5.0 h.

[0071] Example 5: Purification of crude peptide ganirelix

[0072] Sample processing

[0073] (1) Dissolution of crude peptide: Add glacial acetic acid to the crude peptide solution of Example 4.5, with the volume ratio of crude peptide solution to glacial acetic acid being 4:1, and dissolve by ultrasonication.

[0074] (2) Filtration: The dissolved solution is filtered through a 0.45 μm microporous filter membrane, and the filtrates are combined to obtain the crude peptide sample to be purified.

[0075] HPLC purification

[0076] (1) First step purification

[0077] ①First step purification chromatographic conditions:

[0078] Mobile phase: Phase A1: 1‰ trifluoroacetic acid solution; Phase B: pure acetonitrile

[0079] Novasep LC150 chromatography system, chromatographic column filled with C18 10μm chromatographic filler (column efficiency of each batch for the first use is not less than 3000 theoretical plates)

[0080] Monitoring wavelength: 280nm

[0081] Flow rate: 500ml / min

[0082] Purification gradient:Purification gradient:

[0083] ②First step purification process

[0084] Before each injection of purification, a crude peptide sample solution containing 20±5 g of the crude peptide of Example 4.5 was measured, and then diluted with purified water and the crude peptide sample solution according to a volume ratio of 1:1, and HPLC purification was performed. Fractions were collected according to the chromatographic peaks, and the collected fractions were detected by an analytical liquid chromatograph. The fractions with a purity of ≥98.0% and a single impurity of ≤0.5% were qualified fractions in the first step, and the rest were unqualified fractions. The unqualified fractions with a purity of ≥80% were recovered and purified, and the fractions with a purity of <80% were treated as waste liquid.

[0085] The qualified fractions were subjected to rotary evaporation at 30±2°C to remove 30%-50% of the total volume to remove most of the organic solvents, and then the fractions were prepared for the next step of purification.

[0086] (2) Second step purification

[0087] ① Second step purification chromatographic conditions:

[0088] Mobile phase: A2: 50mmol / L sodium perchlorate monohydrate solution, adjusted to pH 2.50±0.05 with phosphoric acid

[0089] B: Pure acetonitrile

[0090] Novasep LC150 chromatography system, chromatographic column filled with C18 10μm chromatographic filler for the preparation of ganirelix acetate

[0091] Monitoring wavelength: 280nm

[0092] Flow rate: 500ml / min

[0093] Purification gradient:

[0094]

[0095] ②The second purification process

[0096] Quantify the qualified fractions in the first step. Perform HPLC purification according to the above chromatographic conditions with a sample load of 10-15 g / time, collect fractions according to the chromatographic peaks, and analyze and test according to the central control detection method. The qualified fractions in the second step are those with a purity of ≥99% and a single impurity of ≤0.15%, and the rest are unqualified fractions. The unqualified fractions with a purity of ≥90% are further purified and recovered in the second step, those with a purity of 80%≤purity<90% are recovered and purified in the first step, and those with a purity of <80% are treated as waste liquid.

[0097] The qualified fractions were subjected to rotary evaporation at 30±2°C to remove 25%-30% of the total volume to remove part of the organic solvent, and then waited for the next step of purification. Sample precipitation should be avoided during rotary evaporation, otherwise the product precipitated and difficult to dissolve.

[0098] Salt transfer purification

[0099] (1) Salt transfer purification chromatographic conditions:

[0100] Mobile phase, A3: 0.2% acetic acid solution by volume, pH adjusted to 6.2-6.5 with aqueous ammonia A4: 0.2% acetic acid solution by volume

[0101] B: Pure acetonitrile

[0102] Novasep LC150 chromatography system, chromatographic column filled with C18 10μm chromatographic packing

[0103] Detection wavelength: 280nm

[0104] Flow rate: 500ml / min

[0105] gradient:

[0106] (2) Salt conversion process

[0107] ① Quantify the qualified fractions in the second step. According to the above chromatographic conditions, perform salt conversion purification with a sample load of 10-15g / time, collect fractions according to the chromatographic peaks, and then analyze them with an analytical liquid chromatograph. Determine the purity of each fraction and the percentage of the single maximum impurity according to the chromatogram peak area normalization method. The parts with purity ≥99.0% and single impurity ≤0.15% are combined and sampled by QA, and sent to the detection of ions. The qualified ion part is the refined peptide solution, which enters the next step of rotary evaporation; the unqualified ions and purity fractions are re-purified by salt conversion.

[0108] Rotary evaporation of peptide solution

[0109] The refined peptide solution was concentrated by rotary evaporation at a water temperature of 30±2°C, concentrated to a volume of 20 ml and then freeze-dried to obtain 1.63 g of white powdery solid refined peptide with a purity of 99.53%. The single small impurity was less than 0.1%. The purification yield was 52% and the total yield was 42%.

[0110] After the purification of ganirelix acetate, the MS of the refined peptide was 1569.604 (M+1). The HPLC spectrum of the refined peptide was as follows: Figure 3 As shown, the target peak of the product is at a retention value of T=17.471 minutes, and the purity is 99.53%.

[0111] In summary, compared with the method of obtaining ganirelix by reacting a linear peptide precursor with ethylaminoethyliminomethanesulfonic acid adopted in patent CN104844694A, the present invention does not need to continuously monitor the pH value in the linear peptide modification reaction, and add alkali solution to maintain the alkalinity of the reaction solution, and the reaction operation is simple; the time used for the modification of the linear peptide reaction in the present invention is 4-6h, and the time used for this step in patent CN 104844694A is 8.0~48.0h, and the linear peptide modification reaction time is short. The purity of the crude ganirelix peptide obtained by the present invention is 85%, and the total yield of the purified refined peptide is 42%, which is higher than the purity of the crude ganirelix peptide of 76% and the total yield of the refined peptide of 34% in patent CN104844694A.

[0112] The above description is only a specific implementation mode of the present invention, not all implementation modes. Any equivalent changes made to the technical solution of the present invention by ordinary technicians in this field after reading the specification of the present invention are covered by the claims of the present invention.

Claims

1. A method for preparing ganirelix acetate, characterized in that: The following steps are involved: 1) amino resin is coupled with amino acid to obtain linear peptide resin; 2) subjecting the linear peptide resin obtained in 1) to acetylation reaction; 3) cleaving the linear peptide resin obtained after the acetylation reaction in 2) to obtain a linear peptide; 4) modifying the linear peptide in 3) with ethylaminoethylimino-1H-pyrazole under alkaline conditions to obtain crude ganirelix acetate peptide; The method described in 4) for modifying the linear peptide in 3) with ethylaminoethylimino-1H-pyrazole under alkaline conditions to obtain crude ganirelix acetate peptide is specifically as follows: the linear peptide in 3) and ethylaminoethylimino-1H-pyrazole are added to water and dissolved, and then the pH is adjusted to 8.5-10.5 for reaction, the reaction time is 4-6 hours, and the reaction temperature is 20°C-30°C.

2. The method for preparing ganirelix acetate according to claim 1, characterized in that: 1) The amino resin includes Rink Amide resin, Rink Amide AM resin or Rink Amide MBHA resin; The substitution degree of the amino resin in 1) is 0.4-1.2 mmol / g.

3. The method for preparing Ganirelix acetate according to claim 2, characterized in that: The amino resin in 1) is Rink Amide resin; the substitution degree of the amino resin in 1) is 0.6-1.0 mmol / g.

4. The method for preparing Ganirelix acetate according to claim 1, characterized in that: 1) The preparation method of the linear peptide resin is: a. Add the amino resin to the solid phase reaction column, swell it with a solvent, remove the Fmoc protecting group and then wash it; b. Dissolve Fmoc-D-Ala-OH and a coupling agent in a solvent and activate them, then add them together to a solid phase reaction column, carry out coupling reaction at 20°C-30°C for 1-3 hours, and wash after the reaction; then remove the Fmoc protecting group and wash; repeat the above process, and sequentially couple amino acids Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-Pal-OH, Fmoc-D-Cpa-OH, and Fmoc-D-Nal-OH from C-terminus to N-terminus.

5. The method for preparing Ganirelix acetate according to claim 4, characterized in that: The temperature of the coupling reaction in 1) is 25°C.

6. The method for preparing Ganirelix acetate according to claim 4, characterized in that: The coupling reaction time in 1) is 2 hours.

7. The method for preparing Ganirelix acetate according to claim 4, characterized in that: The solvent used for the swelling in 1) is one of N,N-dimethylformamide, N-methyl-2-pyrrolidone and dichloromethane.

8. The method for preparing Ganirelix acetate according to claim 7, characterized in that: The solvent used for the swelling in 1) is N,N-dimethylformamide.

9. The method for preparing Ganirelix acetate according to claim 4, characterized in that: 1) The solvent used for washing is one of N,N-dimethylformamide, N-methyl-2-pyrrolidone and dichloromethane.

10. The method for preparing ganirelix acetate according to claim 9, characterized in that: The solvent used in the washing in 1) is N,N-dimethylformamide.

11. The method for preparing Ganirelix acetate according to claim 4, characterized in that: The solvent used in the coupling reaction in 1) is one of N,N-dimethylformamide, N-methyl-2-pyrrolidone and dichloromethane.

12. The method for preparing Ganirelix acetate according to claim 11, characterized in that: The solvent used in the coupling reaction in 1) is N,N-dimethylformamide.

13. The method for preparing Ganirelix acetate according to claim 4, characterized in that: 1) wherein the ratio of Fmoc-AA-OH to the synthesis scale is 3-5:1 in terms of molar ratio; the synthesis scale is the mass of the amino resin multiplied by the degree of substitution of the amino resin, wherein Fmoc-AA-OH is Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-Pal-OH, Fmoc-D-Cpa-OH or Fmoc-D-Nal-OH.

14. The method for preparing ganirelix acetate according to claim 13, characterized in that: 1) The molar ratio of Fmoc-AA-OH to the synthesis scale is 4:

1.

15. The method for preparing Ganirelix acetate according to claim 4, characterized in that: The reagent used for removing the Fmoc protecting group is a 20% piperidine / N,N-dimethylformamide solution.

16. The method for preparing Ganirelix acetate according to claim 4, characterized in that: The coupling agent is a combination of DIPCDI and compound A or a combination of DIPEA, compound A and compound B, wherein compound A is selected from HOAt or HOBt, and compound B is selected from PyAOP, PyBOP, HATU, HBTU or TBTU.

17. The method for preparing Ganirelix acetate according to claim 16, characterized in that: The coupling agent is a composition of DIPCDI and compound A.

18. The method for preparing Ganirelix acetate according to claim 16, characterized in that: The ratio of Fmoc-AA-OH to each component in the coupling agent is calculated by molar ratio as Fmoc-AA-OH:DIPCDI:A=1:1.2:1.1 or Fmoc-AA-OH:DIPEA:A:B=1:2.0:1.1:1.0, wherein Fmoc-AA-OH is Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-Pal-OH, Fmoc-D-Cpa-OH or Fmoc-D-Nal-OH.

19. The method for preparing ganirelix acetate according to claim 1, characterized in that: 2) The acetylation reaction of the linear peptide resin obtained in 1) is specifically as follows: after the amino acid coupling is completed, the Fmoc protecting group is removed and then washed, and then a mixed solution of acetic anhydride, pyridine and a solvent is added, and the reaction is carried out at 20° C.-30° C. for 1-3 hours, and the solvent is one of N,N-dimethylformamide, N-methyl-2-pyrrolidone and dichloromethane.

20. The method for preparing ganirelix acetate according to claim 19, characterized in that: 2) The reaction time is 2 hours.

21. The method for preparing Ganirelix acetate according to claim 19, characterized in that: 2) The reaction temperature is 25°C.

22. The method for preparing Ganirelix acetate according to claim 19, characterized in that: 2) The solvent is N,N-dimethylformamide.

23. The method for preparing Ganirelix acetate according to claim 19, characterized in that: 2) In the acetylation reaction of the linear peptide resin obtained in 1), the molar ratio of acetic anhydride, pyridine, solvent and synthesis scale is 15-30:15-30:15-30:1, and the synthesis scale is the mass of the amino resin multiplied by the substitution degree of the amino resin.

24. The method for preparing Ganirelix acetate according to claim 23, characterized in that: The ratio of acetic anhydride, pyridine, solvent and synthesis scale was 20:20:20:1 in molar ratio.

25. The method for preparing Ganirelix acetate according to claim 1, characterized in that: 3) The lysis solution used for the lysis is a mixture of TFA and H2O, the volume ratio of TFA and H2O is 95:5, and the volume weight ratio of the lysis solution to the linear peptide resin obtained after the acetylation reaction in 2) is 6-8mL:1g; the lysis temperature is room temperature and the time is 2-3 hours.

26. The method for preparing Ganirelix acetate according to claim 25, characterized in that: 3) The lysis time is 2.5 hours.

27. The method for preparing Ganirelix acetate according to claim 1, characterized in that: 4) The reaction time is 5 hours.

28. The method for preparing Ganirelix acetate according to claim 1, characterized in that: 4) The reaction temperature is 25°C.

29. The method for preparing Ganirelix acetate according to claim 1, characterized in that: 4) The solution used for pH adjustment is a sodium hydroxide aqueous solution, and the mass fraction of the sodium hydroxide aqueous solution is 5% to 15%.

30. The method for preparing Ganirelix acetate according to claim 29, characterized in that: 4) The mass fraction of the sodium hydroxide aqueous solution is 10%.

31. The method for preparing Ganirelix acetate according to claim 1, characterized in that: The molar ratio of the ethylaminoethylimino-1H-pyrazole to the linear peptide in 3) is 5-15:

1.

32. The method for preparing Ganirelix acetate according to claim 31, characterized in that: The molar ratio of the ethylaminoethylimino-1H-pyrazole to the linear peptide in 3) is 10:

1.

33. The method for preparing Ganirelix acetate according to claim 1, characterized in that: 4) The method further includes the steps of purification and salt conversion of the crude ganirelix peptide.

34. The method for preparing Ganirelix acetate according to claim 33, characterized in that: The purification is HPLC purification.

35. The method for preparing Ganirelix acetate according to claim 34, characterized in that: The HPLC purification is divided into two steps, including the following steps: (1) the first step of purification: using 10 μm C18 as the stationary phase, the mobile phase A1 phase is a trifluoroacetic acid solution with a volume percentage of 1‰, the B phase is pure acetonitrile, and the gradient elution is 95% A1+5% B changing to 45% A1+55% B; (2) Second step purification: 10 μm C18 was used as the stationary phase, the mobile phase A2 was 50 mmol / L sodium perchlorate monohydrate solution, the pH was adjusted to 2.50±0.05 with phosphoric acid, the mobile phase B was pure acetonitrile, and the gradient elution was changed from 95% A2+5% B to 50% A2+50% B; The salt transfer method comprises the following steps: using 10 μm C18 as the stationary phase, the mobile phase A3 phase is an acetic acid solution with a volume percentage of 0.2%, the pH value is adjusted to 6.2-6.5 with ammonia water, the A4 phase is an acetic acid solution with a volume percentage of 0.2%, the B phase is pure acetonitrile, after 95% A3+5% B isocratic elution, the mobile phase type and gradient are changed to 95% A4+5% B for gradient elution, and the gradient elution is changed from 95% A4+5% B to 50% A4+50% B.

36. The method for preparing Ganirelix acetate according to claim 35, characterized in that: The gradient elution in the first step of purification is specifically as follows: the sample is loaded after equilibration for 5 minutes using a 97% A1+3% B mobile phase, the gradient changes to 95% A1+5% B after loading, the gradient elution is 95% A1+5% B changes to 82% A1+18% B within 2 minutes, and then the gradient elution is 82% A1+18% B changes to 68% A1+32% B for 60 minutes, after the above process completes the elution of the sample, the mobile phase gradient changes from 68% A1+32% B to 55% A1+45% B for 6 minutes, and then the mobile phase gradient changes from 55% A1+45% B to 45% A1+55% B for 6 minutes, and finally after 6 minutes the mobile phase gradient returns to the initial 97% A1+3% B, and this process completes the flushing of the chromatographic column; The gradient elution in the second step of purification is specifically as follows: the sample is loaded after equilibration for 5 minutes using a 95% A2 + 5% B mobile phase, and the gradient elution changes from 95% A2 + 5% B to 75% A2 + 25% B within 4 minutes after loading the sample, and then the gradient elution changes from 75% A2 + 25% B to 63% A2 + 37% B within 60 minutes. After the above process completes the elution of the sample, the mobile phase gradient changes from 63% A2 + 37% B to 50% A2 + 50% B within 1 minute, and then the mobile phase gradient remains unchanged at 50% A2 + 50% B within 4 minutes, and finally after 1 minute, the mobile phase gradient returns to the initial 95% A2 + 5% B, and this process completes the flushing of the chromatographic column; The elution in the salt transfer method is specifically as follows: the sample is loaded after balancing with a mobile phase of 95% A3+5% B for 5 minutes, and then the mobile phase gradient is 95% A3+5% B for isocratic elution for 20 minutes, and then the mobile phase type and gradient are changed to 95% A4+5% B for gradient elution, specifically, the gradient elution is changed from 95% A4+5% B to 75% A4+25% B for 10 minutes, and then the gradient elution is changed from 75% A3+25% B to 63% A4+37% B for 30 minutes. After the above process completes the elution and salt transfer of the sample, the mobile phase gradient is changed from 63% A4+37% B to 50% A4+50% B for 5 minutes, and then the mobile phase gradient remains unchanged at 50% A4+50% B within 5 minutes, and finally the mobile phase gradient returns to the initial 95% A3+5% B after 5 minutes, and this process completes the flushing of the chromatographic column.

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