The invention relates to N, Napos; method for preparing ganirelix acetate from-diethyl iodinated formamidine

Abstract

The invention discloses a method for preparing ganirelix acetate from N, N '-diethyl iodide formamidine, and belongs to the technical field of polypeptide synthesis. The invention relates to a method for preparing ganirelix acetate from N, N '-diethyl iodinated formamidine, which comprises the following steps: taking N, N'-diethyl iodinated formamidine as a key intermediate, gradually constructing a peptide chain through a solid-phase synthesis strategy, selectively carrying out guanidination modification on a lysine side chain, and finally cracking and purifying to obtain a high-purity product. The method is characterized in that a cheap and easily available commercial reagent is adopted to generate a high-activity guanidination intermediate in situ, so that efficient and accurate modification under mild conditions is realized, and the problems of acylation side reaction and heavy metal residue in a traditional process are avoided. The method has the characteristics of high reaction selectivity, short process flow, excellent product purity, low production cost and the like, is suitable for industrial large-scale production, and provides a reliable and economical new way for synthesis of ganirelix acetate.

Description

Technical Field

[0001] This invention relates to the field of polypeptide synthesis technology, and more specifically, to a method for preparing ganirielic acetate from N,N'-diethylformamidinium iodide. Background Technology

[0002] Ganirellix Acetate is a synthetic decapeptide gonadotropin-releasing hormone (GnRH) antagonist whose chemical structure contains the specially modified amino acid residue D-HomoArg(Et)2. 6 and HomoArg(Et)2 8 This drug competitively binds to pituitary GnRH receptors, rapidly and reversibly inhibiting the secretion of gonadotropins, and is widely used in controlled ovulation induction therapy (antagonist regimen) in assisted reproductive technologies.

[0003] Sequence: Ac-D-2-Nal 1 -D-Phe(4-Cl) 2 -D-3-Pal 3 -Ser 4 -Tyr 5 -D-HomoArg(Et)2 6 -Leu 7 -HomoArg(Et)2 8 -Pro 9 -D-Ala-NH2 10 structure: Currently, the synthesis of ganirilac acetate mainly faces the technical challenge of introducing specific amino acid residues. Existing methods can be divided into two categories: Direct use of Fmoc-protected HomoArg(Et)2 amino acids: Due to the lack of effective protection of the guanidino groups of Fmoc-HomoArg(Et)2-OH and Fmoc-D-HomoArg(Et)2-OH, side reactions (such as acylation) are prone to occur during solid-phase synthesis, resulting in a decrease in crude peptide purity and difficulty in purification. Moreover, the raw material is expensive, which greatly increases the production cost.

[0004] Post-modification strategy: First, introduce conventional lysine (Lys), then modify the side chain amino group with guanidinolation. Existing patents (such as CN104231055A and CN113929751A) have reported the use of reagents such as ethylaminoethyliminomethanesulfonic acid and ethylaminoethylimino-1H-pyrazole for modification, but the commercial supply of these reagents is insufficient to meet the needs of industrial production. In addition, CN104017058A uses an N,N'-diethylcarbodiimide and ytterbium trifluoroacetate system, but the introduction of the heavy metal ytterbium increases the risk of quality control problems.

[0005] In summary, the existing technology has the following drawbacks: Poor availability of raw materials: The lack of commercially available sources for special modification reagents restricts large-scale production; High process complexity: Some methods involve toxic metals or unstable intermediates, increasing operational risks; Low cost-effectiveness: Direct use of Fmoc-HomoArg(Et)2-OH makes it difficult to balance yield and purity.

[0006] Therefore, developing a method for preparing ganirielk based on inexpensive and readily available reagents, compatible with solid-phase synthesis processes, and without the involvement of heavy metals is of great significance for promoting its industrial production. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing ganirielic acetate from N,N'-diethylformamidinium iodide, which has the characteristics of high reaction selectivity, short process flow, high product purity and low production cost, and is suitable for large-scale industrial production, providing a reliable and economical new route for the synthesis of ganirielic acetate.

[0008] A method for preparing ganirilac acetate from N,N'-diethylformamidinium iodide includes the following steps: S1. Use amino resin Rink amide AM Resin or Rink amide MBHA Resin with a substitution degree of 0.3~1.5 mmol / g as the starting resin. The amino resin needs to be swollen with DMF solution for 50-70 minutes, and then protected with 20% Pip / DMF solution for 20-40 minutes. Wash and filter to obtain the resin to be reacted. S2. Obtain the ganirilac peptide resin precursor by stepwise coupling via Fmoc solid-phase method: Dissolve Fmoc-D-Ala-OH and HOBt in DMF and cool to 0-15℃. Then, slowly add DIC to the DMF solution while stirring. The molar ratio of Fmoc-D-Ala-OH, HOBt, and DIC is 1:1-2:1-2, and the volume ratio of DMF to Fmoc-D-Ala-OH is 1-2:1. Stir and react at 0-15℃ for 10-20 minutes to obtain an activated Fmoc-D-Ala solution. Add all of this solution to the resin to be reacted in step S1 and couple at 25-35℃ for 120-300 minutes. Filter and wash to obtain Fmoc-D-Ala-amino resin. Use the same method as above to sequentially couple... Protected amino acids Fmoc-Pro-OH, Fmoc-Lys(X)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(X)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3-Pal-OH, Fmoc-D-Phe(4-Cl)-OH, and Fmoc-D-2-Nal-OH were introduced. After incorporation, the resin was protected with 20% Pip / DMF solution for 30 min. The amount of 20% Pip / DMF solution used was 8-20 mL / g of resin. After washing and filtration, the resin to be acetylated was obtained. An acetylation reagent was added, and the reaction was carried out at 20-30℃ for 20-40 min. After washing and filtration, the ganirilac peptide resin precursor Ac-D-2-Nal was obtained. 1 -D-Phe(4-Cl) 2 -D-3-Pal 3 -Ser(tBu) 4 -Tyr(tBu) 5 -D-Lys(X) 6 -Leu 7 -Lys(X) 8 -Pro 9 -D-Ala 10 -Amino resin, wherein X = one of Mmt, Mtt, Alloc, Dde, ivDde; S3, Selective removal of D-Lys 6 and Lys 8 The side chain protecting group X; S3.1 If X is Mmt or Mtt, use 0.5~5% TFA / DCM solution for deprotection, and the amount of deprotection reagent is 6-12 mL / g resin; S3.2 If X is Alloc, deprotection is performed using a DCM solution of 0.1 eq tetraphenylphosphine palladium / 10 eq benzenesilane (eq is based on the amount of amino resin as the reference equivalent), and the amount of deprotection reagent used is 6-12 mL / g resin. S3.3 If X is Dde or ivDde, use 1%~4% hydrazine / DMF solution for deprotection, and the amount of deprotection reagent is 6-12 mL / g resin; S4. Diethylthiourea, triphenylphosphine, iodine and triethylamine are dissolved in DCM in proportion and reacted at 0~10℃ for 10~30 minutes to generate N,N'-diethylformamidinium iodide solution; S5. Mix the resin obtained in step S3 with the N,N'-diethylformamidine iodide solution obtained in step S4, place it in a nitrogen atmosphere, and react at 20~35℃ for 1~3 hours to complete the guanidinization reaction and obtain ganirilactide resin. S6. Using a TFA / TIS / EDT / H2O mixed solution as the lysis reagent, the mixture was lysed at 20~30℃ for 2~4 hours. The reaction mixture was filtered using a sintered funnel, and the filtrate was collected. The resin was washed three times with a small amount of TFA. The filtrates were combined and MTBE was added to precipitate the resin. The precipitate was then washed three times with MTBE and dried to obtain crude ganirilac peptide. S7. The crude peptide was added to water and stirred until completely dissolved. Then it was filtered through a 0.45 μm mixed microporous membrane. The filtrate was purified by reverse-phase preparative chromatography, converted to acetate, and freeze-dried to obtain pure ganirilac acetate.

[0009] Preferably, in step S1, the amount of DMF solution used is 8-12 mL / g resin, and the amount of 20% Pip / DMF solution used is 10-14 mL / g resin.

[0010] Preferably, in step S2, the acetylation reagent is a DMF solution of acetic anhydride and DIEA, with a volume ratio of acetic anhydride, DIEA, and DMF of 3~7:3~7:94~86, preferably acetic anhydride / DIEA / DMF of 5:5:90, and the amount of acetylation reagent used is 2~5 ml / gram of resin.

[0011] Preferably, in step S4, diethylthiourea, triphenylphosphine, iodine and triethylamine are dissolved in DCM in a molar ratio of 1:1~3:1~3:1~2, preferably ethylthiourea, triphenylphosphine, iodine and triethylamine = 1:1:1:1, and the volume ratio of DCM to diethylthiourea is 0.5~3.0:1.

[0012] Preferably, in step S5, the amount of N,N'-diethylformamidinium iodide solution used is 3~10 ml / g resin.

[0013] Preferably, the volume ratio of the TFA / TIS / EDT / H2O mixed solution in step S6 is 85~95:1~5:1~5:1~5, and more preferably TFA / TIS / EDT / H2O=90:5:2.5:2.5, with a dosage of 5~10mL / gram of resin.

[0014] Preferably, the amount of water used in step S7 is 40~60 ml / gram of crude peptide.

[0015] Preferably, the purity of the acetic acid ganirielic acid is ≥99.0%, and the maximum single impurity is ≤0.1%.

[0016] An intermediate for the preparation of ganirilac acetate as described in claim 1, having the structure Ac-D-2-Nal 1 -D-Phe(4-Cl) 2 -D-3-Pal 3 -Ser(tBu) 4 -Tyr(tBu) 5 -D-Lys(X) 6 -Leu 7 -Lys(X) 8 -Pro 9 -D-Ala 10 -Amino resin, wherein X = one of Mmt, Mtt, Alloc, Dde, ivDde.

[0017] Through these two reaction steps, D-Lys is efficiently and rapidly converted into D-Lys 6 and Lys 8 Transformed into D-HomoArg(Et)2 6 and HomoArg(Et)2 8 At the same time, it leverages the advantages of solid-phase peptide synthesis technology in the field of separation and purification, greatly improving reaction efficiency.

[0018] Compared with the prior art, the advantages of this invention are: (1) High purity and low impurities The core innovation of this invention lies in using N,N'-diethylformamidinium iodide as a key active intermediate for guanidinolation modification. This reagent exhibits high reaction selectivity, modifying only the lysine side chain amino group (-NH2), thus avoiding the acylation side reactions caused by insufficient guanidinolation protection in traditional methods. Experimental data show that the crude peptide purity can reach 90.05%, the final product purity is ≥99.0%, and the maximum single impurity is ≤0.1%, significantly superior to the route using Fmoc-HomoArg(Et)2-OH (crude peptide purity is only 83.14%, accompanied by 4.55% acylation impurities).

[0019] (2) High efficiency reaction and short process By generating N,N'-diethylformamidinium iodide in situ, this invention enables guanidinolation reactions to be completed under mild conditions (20-35°C) in just 1-3 hours, whereas traditional methods (such as the carbodiimide / ytterbium system) require high temperatures and long reaction times (>50°C, 6-12 hours). Furthermore, this reagent seamlessly integrates with the solid-phase synthesis process, directly modifying the resin and eliminating the complex purification steps of liquid-phase reactions, thus increasing the overall yield to ≥45% (compared to the low yield problem of traditional methods).

[0020] (3) Raw materials are readily available and cost advantage All raw materials (diethylthiourea, triphenylphosphine, iodine, etc.) are commercially available and inexpensive reagents, avoiding the supply difficulties of custom-modified reagents (such as ethylaminoethyliminomethanesulfonic acid) in existing technologies. At the same time, there is no need to use expensive heavy metal catalysts (such as ytterbium trifluoroacetate), which reduces production costs and meets the impurity control requirements for pharmaceutical production (ICH Q3D).

[0021] (4) Process robustness and scalability This invention employs mature Fmoc solid-phase synthesis technology, combined with optimized protecting group strategies (such as selective removal of Dde / Mtt), to ensure a stable and controllable reaction process. Examples show that this process can be successfully scaled up to a hundred-gram scale (e.g., using 552g of resin in Example 9), with consistent product quality, meeting the requirements of industrial production.

[0022] (5) Green and safety features The entire process involves no heavy metals, and the EDT (ethylene dithiol) in the pyrolysis solution effectively inhibits oxidation side reactions and reduces the generation of toxic waste. Compared with existing technologies, this method is more in line with green chemistry principles and reduces safety risks during production. Attached Figure Description

[0023] Figure 1 This is a process flow diagram of a method for preparing nirexic acetate according to the present invention; Figure 2 It is the process by which diethylthiourea, triphenylphosphine, iodine and triethylamine react to produce N,N'-diethylformamidinium iodide; Figure 3 It is the process by which N,N'-diethylformamidinium iodide reacts with an amino group to form a guanidine group structure; Figure 4 This is an HPLC chromatogram of crude ganirelix peptide prepared by a method for preparing ganirelix acetate according to the present invention; Figure 5 This is an HPLC chromatogram of nirelix acetate, a method for preparing nirelix acetate according to the present invention; Figure 6This is an MS spectrum of a method for preparing nirelix acetate according to the present invention, and nirelix acetate protamine. Figure 7 This is the HPLC chromatogram of crude ganirelix peptide in Example 9 of the method for preparing ganirelix acetate according to the present invention; Figure 8 This is the HPLC chromatogram of ganirelix acetate peptide in Example 9 of the method for preparing ganirelix acetate according to the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0025] In a specific embodiment of the present invention, the Chinese meanings of the English abbreviations used in the application documents are shown in Table 1. Table 1 English abbreviations Chinese name English abbreviations Chinese name Boc tert-Butoxycarbonyl Tyr Tyrosine Fmoc fluorene methyloxycarbonyl Ac Acetyl tBu tert-butyl 2-Nal 3-(2-Naphthyl)-alanine Alloc Allyloxycarbonyl Phe(4-Cl) 4-Chlorophenylalanine TFA Trifluoroacetic acid 3-Pal 3-(3-pyridyl)-alanine Tis Triisopropylsilane Lys Lysine EDT 1,2-Ethylenedithiol <![CDATA[HomoArg(Et)2]]> N,N'-Diethylarginine Rink Amide MBHA Resin, Rink Amide AM Resin Amino resins Ser Serine HPLC High performance liquid chromatography Leu Leucine DIC N,N'-Diisopropylcarbodiimide Ala alanine HOBt 1-Hydroxybenzotriazole Dde 1-(4,4-Dimethyl-2,6-dioxocyclohexyl-1-ethylene)ethyl DIEA Diisopropylethylamine Pro proline DMF N,N-Dimethylformamide IvDde 1-(4,4-Dimethyl-2,6-dioxocyclohexylmethylene)-3-methylbutyl DCM dichloromethane Mmt Methoxytriphenylmethyl 20% Pip / DMF 20% piperidine / N,N-dimethylformamide Mtt Methyltriphenylmethyl The invention will be further illustrated below with examples.

[0026] Example 1: Take 150 mmol Fmoc-D-Ala-OH and 150 mmol HOBt, dissolve them in 150 ml DMF and cool to 10 °C. Take another 150 mmol DIC and slowly add it to the above DMF solution with stirring. Stir and react for 10 minutes at 10 °C to obtain the activated Fmoc-D-Ala solution for later use.

[0027] Take 50 mmol of amino resin (degree of substitution 0.85 mmol / g), swell it with 500 ml of DMF solution for 60 minutes, protect it with 600 ml of 20% Pip / DMF solution for 30 minutes, wash and filter to obtain the resin to be reacted.

[0028] The entire Fmoc-D-Ala activation solution was added to the resin to be reacted, and the coupling reaction was carried out at 30°C for 210 minutes. After filtration and washing, Fmoc-D-Ala-amino resin was obtained.

[0029] Using the same method described above, the protecting amino acids Fmoc-Pro-OH, Fmoc-Lys(Dde)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(Dde)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3-Pal-OH, Fmoc-D-Phe(4-Cl)-OH, and Fmoc-D-2-Nal-OH from the main chain of the table above were sequentially added. After addition, the resin was protected with 600 ml of 20% Pip / DMF solution for 30 min. After washing and filtration, the resin to be acetylated was obtained. Then, 150 ml of acetic anhydride / DIEA / DMF = 5:5:90 solution was added and reacted for 30 min. After washing and filtration, the ganirilac peptide resin precursor Ac-D-2-Nal was obtained. 1 -D-Phe(4-Cl) 2 -D-3-Pal 3 -Ser(tBu) 4 -Tyr(tBu) 5 -D-Lys(X) 6 -Leu 7 -Lys(X) 8 -Pro 9 -D-Ala 10 -Amino resin.

[0030] Example 2: Dissolve 100 mmol of diethylthiourea and 100 mmol of triphenylphosphine in 150 ml of DCM and cool to 5 °C. Separately, add 100 mmol of iodine and 100 mmol of triethylamine to the above DCM solution with stirring. Stir and react for 10 minutes at 10 °C to obtain N,N'-diethylformamidinium iodide solution for later use.

[0031] Example 3: 600 ml of 2% hydrazine / DMF solution was added to the ganiriplastic peptide resin precursor obtained in Example 1. After reacting for 30 min, the D-Lys6 and Lys8 side chain protecting groups Dde were removed. After washing and filtration, the N,N'-diethylformamidinium iodide solution obtained in Example 2 was added to the above resin and reacted at 30°C for 2 h to obtain 150 g of ganiriplastic peptide resin.

[0032] Example 4: Take 300 mmol Fmoc-D-Ala-OH and 300 mmol HOBt, dissolve them in 300 ml DMF and cool to 10 °C. Take another 300 mmol DIC and slowly add it to the above DMF solution with stirring. Stir and react for 10 minutes at 10 °C to obtain an activated Fmoc-D-Ala solution for later use.

[0033] Take 100 mmol of amino resin (degree of substitution 0.90 mmol / g), swell it with 1000 ml of DMF solution for 60 minutes, protect it with 1200 ml of 20% Pip / DMF solution for 30 minutes, wash and filter to obtain the resin to be reacted.

[0034] The entire Fmoc-D-Ala activation solution was added to the resin to be reacted, and the coupling reaction was carried out at 30°C for 210 minutes. After filtration and washing, Fmoc-D-Ala-amino resin was obtained.

[0035] Using the same method described above, the protecting amino acids Fmoc-Pro-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(Alloc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3-Pal-OH, Fmoc-D-Phe(4-Cl)-OH, and Fmoc-D-2-Nal-OH from the main chain of the above table were sequentially added. After addition, the resin was protected with 1200 ml of 20% Pip / DMF solution for 30 min. After washing and filtration, the resin to be acetylated was obtained. Then, 300 ml of acetic anhydride / DIEA / DMF = 5:5:90 solution was added and reacted for 30 min. After washing and filtration, the ganirilactide peptide resin precursor Ac-D-2-Nal was obtained. 1 -D-Phe(4-Cl) 2 -D-3-Pal 3 -Ser(tBu) 4 -Tyr(tBu) 5 -D-Lys(X) 6 -Leu 7 -Lys(X) 8 -Pro 9 -D-Ala 10 -Amino resin.

[0036] Example 5: Dissolve 200 mmol of diethylthiourea and 200 mmol of triphenylphosphine in 300 ml of DCM and cool to 5 °C. Separately, add 200 mmol of iodine and 200 mmol of triethylamine to the above DCM solution with stirring. Stir and react for 10 minutes at 10 °C to obtain N,N'-diethylformamidinium iodide solution for later use.

[0037] Example 6: A solution containing 0.1 eq tetra-triphenylphosphine palladium, 10 eq phenylsilane and 400 ml DCM was added to the ganiritracin peptide resin precursor obtained in Example 4. The reaction was carried out for 3 h to remove the Alloc side chain protecting groups at the D-Lys6 and Lys8 sites. After washing and filtration, the N,N'-diethylformamidinium iodide solution obtained in Example 5 was added to the above resin and reacted at 30°C for 2 h to obtain 295 g of ganiritracin peptide resin.

[0038] Example 7: Take 450 mmol Fmoc-D-Ala-OH and 450 mmol HOBt, dissolve them in 450 ml DMF and cool to 10 °C. Take another 450 mmol DIC and slowly add it to the above DMF solution with stirring. Stir and react for 10 minutes at 10 °C to obtain an activated Fmoc-D-Ala solution for later use.

[0039] Take 150 mmol of amino resin (degree of substitution 1.0 mmol / g), swell it with 1500 ml of DMF solution for 60 minutes, protect it with 1800 ml of 20% Pip / DMF solution for 30 minutes, wash and filter to obtain the resin to be reacted.

[0040] The entire Fmoc-D-Ala activation solution was added to the resin to be reacted, and the coupling reaction was carried out at 30°C for 210 minutes. After filtration and washing, Fmoc-D-Ala-amino resin was obtained.

[0041] Using the same method described above, the protecting amino acids Fmoc-Pro-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(Mtt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3-Pal-OH, Fmoc-D-Phe(4-Cl)-OH, and Fmoc-D-2-Nal-OH from the main chain of the table above were sequentially added. After addition, the resin was protected with 1800 ml of 20% Pip / DMF solution for 30 min. After washing and filtration, the resin to be acetylated was obtained. Then, 450 ml of acetic anhydride / DIEA / DMF = 5:5:90 solution was added and reacted for 30 min. After washing and filtration, the ganirilactide peptide resin precursor Ac-D-2-Nal was obtained. 1 -D-Phe(4-Cl) 2 -D-3-Pal 3 -Ser(tBu) 4 -Tyr(tBu) 5 -D-Lys(X) 6 -Leu 7 -Lys(X) 8-Pro 9 -D-Ala 10 -Amino resin.

[0042] Example 8: Dissolve 300 mmol of diethylthiourea and 300 mmol of triphenylphosphine in 450 ml of DCM and cool to 5 °C. Separately, add 300 mmol of iodine and 300 mmol of triethylamine to the above DCM solution with stirring. Stir and react for 10 minutes at 10 °C to obtain N,N'-diethylformamidinium iodide solution for later use.

[0043] Example 9: 1800 ml of 0.5% TFA / DCM solution was added to the ganiriplastic peptide resin precursor obtained in Example 7, and the reaction was carried out for 30 min to remove the Mtt side chain protecting groups at D-Lys6 and Lys8 sites. After washing and filtration, the N,N'-diethylformamidinium iodide solution obtained in Example 8 was added to the above resin, and the reaction was carried out at 30°C for 2 h to obtain 402 g of ganiriplastic peptide resin.

[0044] Example 10: The ganiriplastic peptide resin obtained in Example 3 was added to a TFA mixed solution with a ratio of TFA / TIS / EDT / H2O of 90:5:2.5:2.5. 6 ml of lysis reagent was added per gram of resin. The mixture was stirred at 25°C for 3 hours. The reaction mixture was filtered using a sintered glass funnel, and the filtrate was collected. The resin was washed three times with a small amount of TFA. The filtrates were combined, and 8 times the volume of MTBE was added to precipitate the resin. The precipitate was then washed three times with MTBE and dried to obtain a white powder, which was 80.6 g of crude ganiriplastic peptide with a purity of 90.05%. Figure 4 ).

[0045] Example 11: Take the crude ganiriplastic product obtained in Example 10, add water and stir until completely dissolved. Use 50 ml of water for every gram of crude ganiriplastic product. Filter the crude peptide solution through a 0.45 μm mixed microporous membrane.

[0046] Purification was performed using reversed-phase preparative chromatography, and qualified fractions were collected. The fractions were combined, converted to acetate, and concentrated under reduced pressure to obtain an aqueous solution of ganirilac peptide. This solution was freeze-dried to yield 38.69 g of ganirilac peptide acetate with a purity of 99.46% and a maximum single impurity of 0.08%. Figure 2 The overall yield was 49.3%, and the molecular weight was 1571.05 [M+H]. + This is consistent with the theoretical molecular weight of 1570.34. Figure 5 ).

[0047] Example 12: This comparative example directly uses Fmoc-HomoArg(Et)2-OH and Fmoc-D-HomoArg(Et)2-OH for coupling.

[0048] Take 150 mmol Fmoc-D-Ala-OH and 150 mmol HOBt, dissolve them in 150 ml DMF and cool to 10 °C. Take another 150 mmol DIC and slowly add it to the above DMF solution with stirring. Stir and react for 10 minutes at 10 °C to obtain an activated Fmoc-D-Ala solution for later use.

[0049] Take 50 mmol of amino resin (degree of substitution 0.85 mmol / g), swell it with 500 ml of DMF solution for 60 minutes, protect it with 600 ml of 20% Pip / DMF solution for 30 minutes, wash and filter to obtain the resin to be reacted.

[0050] The entire Fmoc-D-Ala activation solution was added to the resin to be reacted, and the coupling reaction was carried out at 30°C for 210 minutes. After filtration and washing, Fmoc-D-Ala-amino resin was obtained.

[0051] Using the same method described above, the protecting amino acids Fmoc-Pro-OH, Fmoc-HomoArg(Et)2-OH, Fmoc-Leu-OH, Fmoc-D-HomoArg(Et)2-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3-Pal-OH, Fmoc-D-Phe(4-Cl)-OH, and Fmoc-D-2-Nal-OH were sequentially added to the main chain of the main chain listed in the table above. After addition, the resin was protected with 600 ml of 20% Pip / DMF solution for 30 min. After washing and filtration, the resin to be acetylated was obtained. Then, 150 ml of acetic anhydride / DIEA / DMF = 5:5:90 solution was added and reacted for 30 min. After washing and filtration, the ganirilac peptide resin Ac-D-2-Nal was obtained. 1 -D-Phe(4-Cl) 2 -D-3-Pal 3 -Ser(tBu) 4 -Tyr(tBu) 5 -D-HomoArg(Et)2 6 -Leu 7 -HomoArg(Et)2 8 -Pro 9 -D-Ala 10 -Amino resin.

[0052] Take the above-mentioned ganiriplastic peptide resin and add it to a TFA mixed solution with a ratio of TFA / TIS / EDT / H2O = 90:5:2.5:2.5. The lysis reagent is 6 ml / g resin. Stir the reaction mixture at 25°C for 3 hours. Filter the reaction mixture using a sintered glass funnel, collect the filtrate, and wash the resin three times with a small amount of TFA. Combine the filtrates and add 8 times the volume of MTBE to precipitate the resin. Wash the precipitate three times with MTBE, and dry it to obtain a white powder, which is 75.3 g of crude ganiriplastic peptide with a purity of 83.14%. Figure 6 ).

[0053] The same purification process as in Example 11 was used. The distillate was collected, converted to acetate, and concentrated under reduced pressure to obtain an aqueous solution of ganirelix peptide. This solution was then freeze-dried to obtain ganirelix peptide acetate with a purity of 98.14% and a maximum single impurity of 0.42%. Figure 7 ).

[0054] Performance testing Based on the HPLC chromatogram results of the above embodiments, The test data is shown in the table below: Finally, it should be noted that the above embodiments are only used to clearly illustrate the specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. Under the premise of the core concept of the present invention (using N,N'-diethylformamidinium iodide as a key intermediate, to prepare ganirilac acetate through solid-phase synthesis and selective guanidinolation modification), any person skilled in the art can make appropriate adjustments or equivalent substitutions to the raw material ratios, protecting group types, reaction temperatures, and purification conditions. These reasonable changes or modifications based on the essential principles of the present invention should all be considered to fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing ganirilac acetate from N,N'-diethylformamidinium iodide, characterized in that, Includes the following steps: S1. Use amino resins Rink amide AM Resin or Rink amide MBHA Resin with a degree of substitution of 0.3~1.5 mmol / g as starting resins. The amino resins need to be swollen with DMF solution for 50-70 minutes, and then protected with 20% Pip / DMF solution for 20-40 minutes. After washing and filtration, the resin to be reacted is obtained. S2. Obtain the ganirilac peptide resin precursor by stepwise coupling via Fmoc solid-phase method: Dissolve Fmoc-D-Ala-OH and HOBt in DMF and cool to 0-15℃. Then, slowly add DIC to the DMF solution while stirring. The molar ratio of Fmoc-D-Ala-OH, HOBt, and DIC is 1:1-2:1-2, and the volume ratio of DMF to Fmoc-D-Ala-OH is 1-2:

1. Stir and react at 0-15℃ for 10-20 minutes to obtain an activated Fmoc-D-Ala solution. Add all of this solution to the resin to be reacted in step S1 and couple at 25-35℃ for 120-300 minutes. Filter and wash to obtain Fmoc-D-Ala-amino resin. Use the same method as above to sequentially couple... Protected amino acids Fmoc-Pro-OH, Fmoc-Lys(X)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(X)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3-Pal-OH, Fmoc-D-Phe(4-Cl)-OH, and Fmoc-D-2-Nal-OH were introduced. After incorporation, the resin was protected with 20% Pip / DMF solution for 30 min. The amount of 20% Pip / DMF solution used was 8-20 mL / g of resin. After washing and filtration, the resin to be acetylated was obtained. An acetylation reagent was added, and the reaction was carried out at 20-30℃ for 20-40 min. After washing and filtration, the ganirilac peptide resin precursor Ac-D-2-Nal was obtained. 1 -D-Phe(4-Cl) 2 -D-3-Pal 3 -Ser(tBu) 4 -Tyr(tBu) 5 -D-Lys(X) 6 -Leu 7 -Lys(X) 8 -Pro 9 -D-Ala 10 -Amino resin, wherein X = one of Mmt, Mtt, Alloc, Dde, ivDde; S3, Selective removal of D-Lys 6 and Lys 8 The side chain protecting group X; S3.1 If X is Mmt or Mtt, use 0.5~5% TFA / DCM solution for deprotection, and the amount of deprotection reagent is 6-12 mL / g resin; S3.2 If X is Alloc, deprotection is performed using a DCM solution of 0.1 eq tetraphenylphosphine palladium / 10 eq benzenesilane (eq is based on the amount of amino resin as the reference equivalent), and the amount of deprotection reagent used is 6-12 mL / g resin. S3.3 If X is Dde or ivDde, use 1%~4% hydrazine / DMF solution for deprotection, and the amount of deprotection reagent is 6-12 mL / g resin; S4. Diethylthiourea, triphenylphosphine, iodine and triethylamine are dissolved in DCM in proportion and reacted at 0~10℃ for 10~30 minutes to generate N,N'-diethylformamidinium iodide solution; S5. Mix the resin obtained in step S3 with the N,N'-diethylformamidine iodide solution obtained in step S4, place it in a nitrogen atmosphere, and react at 20~35℃ for 1~3 hours to complete the guanidinization reaction and obtain ganirilactide resin. S6. Using a TFA / TIS / EDT / H2O mixed solution as the lysis reagent, the mixture was lysed at 20~30℃ for 2~4 hours. The reaction mixture was filtered using a sintered funnel, and the filtrate was collected. The resin was washed three times with a small amount of TFA. The filtrates were combined and MTBE was added to precipitate the resin. The precipitate was then washed three times with MTBE and dried to obtain crude ganirilac peptide. S7. The crude peptide was added to water and stirred until completely dissolved. Then it was filtered through a 0.45 μm mixed microporous membrane. The filtrate was purified by reverse-phase preparative chromatography, converted to acetate, and freeze-dried to obtain pure ganilide acetate.

2. The method for preparing ganirilac acetate from N,N'-diethylformamidinium iodide according to claim 1, characterized in that: In step S1, the amount of DMF solution used is 8-12 mL / g resin, and the amount of 20% Pip / DMF solution used is 10-14 mL / g resin.

3. The method for preparing ganirilac acetate from N,N'-diethylformamidinium iodide according to claim 1, characterized in that: In step S2, the acetylation reagent is a DMF solution of acetic anhydride and DIEA, with a volume ratio of acetic anhydride / DIEA / DMF = 3~7:3~7:94~86, and the amount of acetylation reagent used is 2~5 ml / gram of resin.

4. The method for preparing ganirilac acetate from N,N'-diethylformamidinium iodide according to claim 1, characterized in that: In step S4, diethylthiourea, triphenylphosphine, iodine and triethylamine are dissolved in DCM in a molar ratio of 1:1~3:1~3:1~2, and the volume ratio of DCM to diethylthiourea is 0.5~3.0:

1.

5. The method for preparing ganirilac acetate from N,N'-diethylformamidinium iodide according to claim 1, characterized in that: In step S5, the amount of N,N'-diethylformamidinium iodide solution used is 3~10 ml / g resin.

6. The method for preparing ganirilac acetate from N,N'-diethylformamidinium iodide according to claim 1, characterized in that: In step S6, the volume ratio of the TFA / TIS / EDT / H2O mixed solution is 85~95:1~5:1~5:1~5, and the dosage is 5~10mL / gram of resin.

7. The method for preparing ganirilac acetate from N,N'-diethylformamidinium iodide according to claim 1, characterized in that: In step S7, the amount of water used is 40-60 ml / gram of crude peptide.

8. The method for preparing ganirilac acetate from N,N'-diethylformamidinium iodide according to claim 1, characterized in that: The purity of the ganirielic acetate is ≥99.0%, and the maximum single impurity is ≤0.1%.

9. An intermediate for use in the method of preparing ganirilac acetate as described in claim 1, characterized in that: Its structure is Ac-D-2-Nal 1 -D-Phe(4-Cl) 2 -D-3-Pal 3 -Ser(tBu) 4 -Tyr(tBu) 5 -D-Lys(X) 6 -Leu 7 -Lys(X) 8 -Pro 9 -D-Ala 10 -Amino resin, wherein X = one of Mmt, Mtt, Alloc, Dde, ivDde.

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