Preparation method of ganirelix

Abstract

The invention relates to the field of chemical synthesis of polypeptide drugs, in particular to a preparation method of ganirelix. The preparation method of ganirelix comprises the following steps: by taking amino resin as initial resin, sequentially coupling amino acids by using Lys protected by an Mtt or Mmt side chain; after the coupling is completed, removing an Fmoc protecting group; carrying out acetylation reaction on the N terminal; removing a side chain protecting group of Lys; the method comprises the following steps: carrying out a side chain modification reaction by using diethylamine and a catalyst to convert Lys into (Diet) Har, the catalyst being CDI and copper chloride, or triphosgene and dibromotriphenylphosphine; and finally cracking to obtain ganirelix. According to the preparation method of ganirelix acetate, diethylamine and other cheap materials are used and are all operated on the solid-phase carrier, the operation is simple, the cost is low, and the yield of the obtained ganirelix acetate refined product reaches 80% or above.

Description

[0001] Cross-reference

[0002] This application claims the priority of the patent application with the application number "202410421151.8" and the invention title "A Preparation Method of Ganirelix" filed on April 8, 2024, the content of which is incorporated herein by reference in its entirety. Technical Field

[0003] This application relates to the field of chemical synthesis of polypeptide drugs. Specifically, it relates to a preparation method of ganirelix. Background Art

[0004] Ganirelix is an antagonist of gonadotropin-releasing hormone (GnRH), which can competitively block the GnRH receptor on pituitary gonadotroph cells and the subsequent transduction pathway, thereby producing a rapid and reversible inhibitory effect on gonadotropin secretion. Its inhibitory effect on luteinizing hormone (LH) secretion by the pituitary gland is stronger than that on follicle-stimulating hormone (FSH). Ganirelix cannot cause the initial release of endogenous gonadotropins, which is consistent with the antagonistic effect. In the controlled ovarian stimulation treatment in assisted reproductive medicine, ganirelix is used in the antagonist protocol, which can reduce the treatment burden of women receiving assisted reproductive technology and also reduce the occurrence of serious adverse events such as ovarian hyperstimulation syndrome (OHSS). For women with ovarian hyperstimulation syndrome, ganirelix can prevent LH fluctuations and related stimulations and increase the implantation and pregnancy rates. Therefore, ganirelix has high medicinal value and broad market prospects.

[0005] The injection of ganirelix acetate was developed and produced by Merck & Co., Inc. It was launched in the United States in 1999 and approved for marketing in China by the China National Food and Drug Administration in 2013 under the trade name ORGALUTRAN.

[0006] Ganirelix is a decapeptide drug with the following amino acid sequence:

[0007] Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-(Diet)Har-Leu-(Diet)Har-Pro-D-Ala-NH2.

[0008] The existing preparation methods of ganirelix mainly include the Boc solid-phase synthesis method and the Fmoc solid-phase synthesis method. The final deprotection of the Boc solid-phase synthesis method mostly uses highly toxic HF method or TFMSA (trifluoromethanesulfonic acid) method, with a large amount of process waste liquid, low yield, high cost, and serious environmental pollution, which is not conducive to industrial production. The Fmoc solid-phase synthesis method mainly includes the fragment synthesis method and the stepwise coupling method.

[0009] 1) Fragment synthesis method:

[0010] Patent CN107056894B1 adopts the synthesis method of fragment [1-5] and [6-10]; using Fmoc-Lys(X)-OH, where X is mtt or mmt, as raw materials, solid-phase synthesis is carried out with amino resin as the carrier. After removing the side-chain protecting group on Lys, Lys is modified with ethylaminoethanimidoyl methanesulfonic acid to obtain the peptide resin fragment A [6-10], and the intermediate fragment B [1-5] is synthesized by the solid-phase method; then the intermediate fragment B [1-5] is connected to the peptide resin fragment A [6-10], and ganirelix is obtained by cleavage. However, the fragment synthesis operation is complex, the cost is 2-3 times that of normal amino acid coupling one by one, and the high-arginine side chain will have side reactions with the fragment.

[0011] Patent application CN114478708A adopts the synthesis method of fragment [1-2] and [3-10]; using Fmoc-(Diet)Har-OH as the raw material, solid-phase synthesis is carried out with amino resin as the carrier to obtain the peptide resin fragment A [3-10], and the intermediate fragment B [1-2] is synthesized by the solid-phase method; then the intermediate fragment B [1-2] is connected to the peptide resin fragment A [6-10], and ganirelix is obtained by cleavage. However, the fragment synthesis operation is complex, the cost is 3 times that of normal condensation; moreover, the synthesis cost of Fmoc-(Diet)Har-OH is high, it is easy to decompose, and the high-arginine side chain will have side reactions with the fragment.

[0012] 2) Step-by-step coupling method:

[0013] Patent application CN102584945A uses Fmoc-(Diet)Har-OH as the raw material for solid-phase synthesis by step-by-step amino acid coupling, but the synthesis cost of Fmoc-(Diet)Har-OH is high, it is easy to decompose, and in the subsequent acetylation reaction, the side chain of high-arginine will have side reactions with acetic anhydride. Patent CN104371010B uses Fmoc-(Diet)Har-OH as the raw material in the step-by-step amino acid coupling of solid-phase synthesis, and finally uses Ac-D-Nal-OH as the raw material for condensation to avoid side reactions of the high-arginine side chain with acetic anhydride; but the synthesis cost of Fmoc-(Diet)Har-OH is high, it is easy to decompose, and the high-arginine side chain will still have side reactions with amino acids. Patent application CN110563812A also uses Fmoc-(Diet)Har-OH as the raw material for step-by-step amino acid coupling in solid-phase synthesis. Then, in the N-terminal acetylation reaction, glacial acetic acid / HOBt / DIC is used to activate for 5-10 min at 0-5 °C in an ice bath and then react with the resin to avoid side reactions of the active group of the high-arginine side chain during acetylation; but the synthesis cost of Fmoc-(Diet)Har-OH is high, it is easy to decompose, and the side reaction of the high-arginine side chain with acetic acid cannot be completely overcome.

[0014] The patent application CN104844694A uses Fmoc-Lys(Boc)-OH as a raw material and conducts solid-phase synthesis by coupling amino acids one by one. After the N-terminal acetylation reaction, the synthesized decapeptide is cleaved from the resin, and then Lys is modified with ethylaminoethanimidamidosulfonic acid in a liquid-phase reaction to obtain ganirelix. This method first conducts solid-phase synthesis and then modifies the Lys side chain in the liquid phase, with complex operations.

[0015] The patent CN104017058B uses Fmoc-Lys(Dde)-OH as a raw material and conducts solid-phase synthesis by coupling amino acids one by one. After the N-terminal acetylation reaction, first, hydrazine hydrate is used to remove the Dde protecting group on the Lys side chain, then ytterbium trifluoromethanesulfonate and DIC are used to modify Lys, and finally, ganirelix is obtained by cleavage. This method uses the highly toxic reagent hydrazine hydrate to remove the protecting group on Lys, causing significant pollution to the environment, and uses ytterbium metal for side-chain modification, with high prices leading to high costs. Similarly, the patent application CN117285600A uses Fmoc-Lys(Alloc)-OH as a raw material for solid-phase synthesis. After the N-terminal acetylation reaction, first, a Pd reagent and an Alloc protecting group capturer are used to remove the Alloc protecting group on the Lys side chain, and then modification reagents such as ethylaminoethylimino-1H-pyrazole are used to modify Lys, and finally, ganirelix is obtained by cleavage. This method uses the precious metal Pd to remove the side-chain protecting group of Lys, with high costs.

[0016] It can be seen that the existing synthesis methods of ganirelix have technical problems such as complex synthesis processes, high costs, and low yields. To solve the above problems, this application provides a preparation method of ganirelix. Summary of the Invention

[0017] This application aims at the deficiencies in the prior art and provides a preparation method of ganirelix, which is simple in operation, low in cost, and high in yield.

[0018] The preparation method of ganirelix described in this application includes the following steps:

[0019] (1) Using amino resin as the starting resin, through the Fmoc solid-phase synthesis method, amino acids are coupled in sequence. When coupling the 6th and 8th amino acids at the N-terminus, Fmoc-D-Lys(X1)-OH and Fmoc-Lys(X2)-OH are used respectively, where X1 and X2 are the same or different and are respectively selected from Mtt or Mmt. When coupling the 4th and 5th amino acids at the N-terminus, Ser and Tyr with N-terminal Fmoc protection and side-chain protection are used respectively; after coupling is completed, the N-terminal Fmoc protecting group is removed;

[0020] (2) Conduct an acetylation reaction on the N-terminus;

[0021] (3) Remove the Mtt and / or Mmt protecting groups from the side chain of Lys;

[0022] (4) Carry out side chain modification reaction using diethylamine and a catalyst to convert the amino acids at positions 6 and 8 on the resin obtained in step (3) into D-(Diet)Har and (Diet)Har respectively. The catalyst is CDI and copper chloride, or triphosgene and dibromotriphenylphosphine;

[0023] (5) Cleave to obtain ganirelix.

[0024] Among them, in step (3), the Mtt and / or Mmt protecting groups are removed using a hexafluoroisopropanol solution; the hexafluoroisopropanol solution is a hexafluoroisopropanol / dichloromethane solution or a hexafluoroisopropanol / dichloroethane solution; preferably a 20 - 40% (V) hexafluoroisopropanol / dichloromethane solution or a 20 - 40% (V) hexafluoroisopropanol / dichloroethane solution, more preferably a 30% (V) hexafluoroisopropanol / dichloromethane solution or a 30% (V) hexafluoroisopropanol / dichloroethane solution. The dosage ratio of the hexafluoroisopropanol solution to the peptide resin complex obtained in step (2) is 10 - 50 mL / mmol resin, preferably 20 - 30 mL / mmol resin. The reaction temperature for the deprotection reaction is 20 - 80°C, preferably 30 - 60°C, such as 30°C, 40°C, 50°C, 60°C, 70°C, and the reaction time for the deprotection reaction is 10 min - 60 min, preferably 20 min - 30 min. The deprotection reaction can be repeated 2 - 4 times, preferably 3 times.

[0025] Specifically, add the hexafluoroisopropanol solution to the reactor for the deprotection reaction, filter after the reaction, repeat the deprotection reaction, filter after the reaction, wash with an organic solvent, and filter.

[0026] Among them, in step (4), the molar ratio of diethylamine to the peptide resin complex obtained in step (3) is 2 - 10:1, preferably 3 - 6:1. The molar ratio of the catalyst CDI, copper chloride to the peptide resin complex obtained in step (3) is (1 - 5):(1 - 3):1, preferably (1 - 3):1:1. The molar ratio of the catalyst triphosgene:dibromotriphenylphosphine to the peptide resin complex obtained in step (3) is (1 - 5):(1 - 3):1, preferably (1 - 3):1:1. The reaction temperature for the side chain modification reaction in step (4) is 20 - 80°C, preferably 30 - 60°C, such as 30°C, 40°C, 50°C, 60°C, 70°C. The reaction time for the side chain modification reaction in step (4) is 0.5 - 3 h, preferably 1 - 2 h. The reaction solvent for the side chain modification reaction in step (4) is selected from one or more of dichloromethane, dichloroethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, and preferably, the reaction solvent is selected from dichloromethane or dichloroethane.

[0027] Specifically, in step (4), the solution obtained by dissolving diethylamine and a catalyst in a reaction solvent is added to the reactor of step (3) to carry out a side-chain modification reaction. After the reaction, filtration is performed, and then an organic solvent is added for washing and filtration; the organic solvent is selected from one or more of dichloromethane, dichloroethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and dimethyl sulfoxide. Preferably, the organic solvent is N,N-dimethylformamide.

[0028] Among them, preferably, X1 and X2 are the same, and more preferably, both X1 and X2 are Mtt.

[0029] Among them, preferably, the resin obtained in step (3) is Ac-D-Nal-D-Cpa-D-Pal-Ser(X3)-Tyr(X4)-D-Lys-Leu-Lys-Pro-D-Ala-NH-resin; the resin obtained in step (4) is Ac-D-Nal-D-Cpa-D-Pal-Ser(X3)-Tyr(X4)-D-(Diet)Har-Leu-(Diet)Har-Pro-D-Ala-NH-resin; X3 and X4 are the same or different and are protecting groups for OH on the side chain; preferably, X3 and X4 are selected from t Bu or Boc; more preferably, X3 and X4 are the same, and most preferably, both X3 and X4 are t Bu.

[0030] Among them, in step (1), the amino resin used is Rink Amide AM resin, Rink Amide resin, RinkAmide MBHA resin, Rink Amide BHA resin, Sieber resin, Sieber AM resin, etc., and the resin substitution degree is 0.3 mmol / g to 1.5 mmol / g, preferably 0.5 mmol / g to 1.0 mmol / g.

[0031] In step (1), the successively coupled amino acids are Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-Lys(X2)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(X1)-OH, Fmoc-Tyr(X4)-OH, Fmoc-Ser(X3)-OH, Fmoc-D-Pal-OH, Fmoc-D-Cpa-OH, Fmoc-D-Nal-OH. The coupling reagent can be a conventional reagent in the field of polypeptide synthesis. In some embodiments, the coupling reagent is DIC / HOBt, DIC / HOAt. The molar ratio of the coupling reagent, the amino acid to be coupled, and the resin or peptide resin complex is 1-5:1-5:1, preferably 2-3:2-3:1. The reaction solvent for the coupling reaction can be selected from one or more of dichloromethane, dichloroethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and dimethyl sulfoxide. Preferably, the reaction solvent is N,N-dimethylformamide. When coupling, the reaction solution of the amino acid to be coupled and the coupling reagent dissolved in the reaction solvent is added to the reactor for the coupling reaction. The reaction temperature of the coupling reaction is 20-80°C, preferably 30-60°C, such as 30°C, 40°C, 50°C, 60°C, 70°C. The reaction time of the coupling reaction is 0.5-3 h, preferably 1-2 h. The amino group on the amino resin is protected by Fmoc. In the successive coupling, each amino acid N-terminus also has an Fmoc protecting group. Therefore, it is necessary to first remove the Fmoc protecting group before coupling. A piperidine solution is used to remove the Fmoc protecting group. Preferably, a piperidine / DMF solution is used, more preferably a 10-30% (V) piperidine / DMF solution, and most preferably a 20% (V) piperidine / DMF solution. The dosage of the piperidine solution and the ratio of the resin or peptide resin complex are preferably 10-50 mL / mmol resin, more preferably 20-30 mL / mmol resin. The reaction temperature of the Fmoc protecting group removal reaction is 20-80°C, preferably 30-60°C, such as 30°C, 40°C, 50°C, 60°C, 70°C. The reaction time of the removal reaction is 10 min-60 min, preferably 20 min-30 min. After each coupling reaction and after the removal reaction, filtration is carried out, followed by washing with an organic solvent and then filtration again. The organic solvent is selected from one or more of dichloromethane, dichloroethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and dimethyl sulfoxide. Preferably, the organic solvent is N,N-dimethylformamide.

[0032] Specifically, in step (1), first add the amino resin into the reactor, and use an organic solvent (selected from one or more of dichloromethane, dichloroethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, preferably N,N-dimethylformamide) to swell and remove the Fmoc protecting group on the resin; add the reaction solution of Fmoc-D-Ala-OH and the coupling reagent in the reaction solvent into the reactor for the coupling reaction; remove the N-terminal Fmoc protecting group; repeat the above process to couple amino acids in sequence from the C-terminal to the N-terminal; finally remove the N-terminal Fmoc protecting group. After resin swelling, after each coupling reaction, and after each deprotection reaction, filter, add an organic solvent for washing, and then filter.

[0033] In step (2), the N-terminal amino group can be acetylated by a conventional acetylation method in the art. Preferably, acetic anhydride and a base are used for the acetylation reaction. The base can be selected from N,N-diisopropylethylamine, pyridine, etc. The molar ratio of acetic anhydride: base: peptide resin complex is 1-5:1-5:1, preferably 2-3:2-3:1. The reaction solvent for the acetylation reaction can be selected from one or more of dichloromethane, dichloroethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide. Preferably, the reaction solvent is dichloromethane. The reaction temperature for the acetylation reaction is 20-80°C, preferably 30-60°C, such as 30°C, 40°C, 50°C, 60°C, 70°C, and the reaction time for the acetylation reaction is 0.5-3 h, preferably 1-2 h.

[0034] Specifically, in step (2), add the reaction solution of acetic anhydride and the base in the reaction solvent into the reactor for the acetylation reaction; after the reaction is completed, filter, wash with an organic solvent, and then filter.

[0035] Among them, in step (5), ganirelix is obtained by cleavage, sedimentation, and purification. The cleavage agent for the cleavage is a mixture of TFA and at least one reagent selected from the following: water, EDT, benzyl methyl sulfide, phenol, TES, TIS, DODT. Preferably, the cleavage agent contains more than 80% TFA, more preferably more than 85% TFA, and more preferably more than 90% TFA. The cleavage agent is preferably a mixture of TFA, EDT, benzyl methyl sulfide, phenol, and water, with a preferred volume ratio of 90:2.5:2.5:2.5:2.5; the dosage ratio of the cleavage agent to the peptide resin complex is 10 - 50 mL / mmol resin, preferably 20 - 30 mL / mmol resin. The cleavage temperature is 0 - 30 °C, preferably 0 - 25 °C, and the cleavage time is 0.5 - 3 h, preferably 1 - 2 h. After cleavage, filtration is carried out, a sedimentation agent is added to the filtrate for precipitation, and the obtained crude product is purified and freeze-dried to obtain ganirelix. The sedimentation agent can be selected from one or more of ether, petroleum ether, methyl tert-butyl ether, isopropyl ether, dipropyl ether, and ethylene glycol dimethyl ether, preferably one or more of ether and methyl tert-butyl ether; the volume ratio of the sedimentation agent to the filtrate is 1:5 - 20, preferably 1:10 - 15.

[0036] Specifically, in step (5), the cleavage agent is added to the reactor for cleavage reaction, filtered, the filtrate is added to the sedimentation agent for precipitation, and the crude product is obtained by centrifugation or filtration and then purified to obtain ganirelix.

[0037] In the method of the present application, the organic solvent for washing is selected from one or more of dichloromethane, dichloroethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and dimethyl sulfoxide. Preferably, the organic solvent is N,N-dimethylformamide.

[0038] The present application also provides a preparation method of ganirelix acetate. After ganirelix is prepared according to the method of the present application, through a salt conversion and an optional freeze-drying (such as lyophilization) step, ganirelix acetate is obtained.

[0039] Through research, it is found that compared with the prior art, the present application has at least the following beneficial effects:

[0040] The preparation method of ganirelix provided by the present application adopts a step-by-step coupling method, uses side-chain protected lysine as the raw material, and after N-terminal acetylation, lysine is modified to (Diet)Har on the solid-phase carrier. On the one hand, it avoids the use of the relatively expensive and easily decomposed amino acid (Diet)Har, and on the other hand, the operation is simple, thus reducing the process cost.

[0041] The present application uses diethylamine and a catalyst to modify lysine on the solid-phase carrier. Diethylamine and the catalyst used are inexpensive, and the raw material cost is low.

[0042] When coupling one by one, lysine protected by Mtt or Mmt is used as a raw material for the side chain. After N-terminal acetylation, the side chain protecting groups Mmt or Mtt can be conveniently and low-costly removed using a hexafluoroisopropanol solution.

[0043] The method for preparing ganirelix provided by the present application uses inexpensive materials such as diethylamine and is all operated on a solid-phase carrier. The operation is simple and the cost is low, and the yield of the obtained high-quality ganirelix acetate reaches more than 80%. Description of the Drawings

[0044] Figure 1 It is a synthetic route diagram of the method for preparing ganirelix described in the present application.

[0045] Figure 2 It is the HPLC chromatogram of the obtained high-quality ganirelix acetate in Example 1.

[0046] Figure 3 It is the MS chromatogram of the obtained high-quality ganirelix acetate in Example 1. Detailed Description of the Invention

[0047] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. And the laboratory operation steps used herein are all conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present application, the definitions and explanations of related terms are provided below.

[0048] "Peptide" has its ordinary meaning in the art and can refer to a compound formed by covalently linking together through an amide bond (peptide bond) formed by the condensation and dehydration of the carboxyl group of one amino acid molecule and the amino group of another amino acid molecule.

[0049] The naming principle used to define peptides is commonly used in the art, where the N-terminal amino group appears on the left and the C-terminal carboxyl group appears on the right.

[0050] "Amino acid" has its ordinary meaning in the art and can include natural amino acids and unnatural amino acids. The abbreviations of natural amino acid residues in the present application are the standard three-letter codes used in the art to refer to one of the 20 common amino acids.

[0051] The suffixes "-OH" and "-NH2" after a hyphen or in parentheses respectively refer to the free acid and amide forms of a polypeptide or an amino acid. For example: Fmoc-Tyr-OH refers to the free acid form of the Tyr amino acid with its N-terminal amino group protected by Fmoc. Peptide-NH2 refers to the amide form of the amino acid at the C-terminal of the peptide chain.

[0052] The peptide resin complex obtained in steps (1)-(4) is a peptide resin complex in which an amide bond is formed between the C-terminal carboxyl group of the peptide and the amino group of the amino resin.

[0053] "Protecting group" or "protective group" has its ordinary meaning in the art. A protecting group includes a chemical moiety that is attached to or configured to be attached to a reactive group (i.e., the protected group) within a molecule (e.g., a peptide) such that the protecting group prevents or otherwise inhibits the protected group from participating in a reaction. Protection can be achieved by attaching the protecting group to the molecule. Deprotection can occur when the protecting group is removed from the molecule, e.g., by a chemical transformation that removes the protecting group.

[0054] When a protecting group is indicated in parentheses after an amino acid, it means that the reactive side chain group of the amino acid is protected by the protecting group in the parentheses. For example, Tyr( t Bu) refers to Tyr in which the side chain reactive group OH is protected as a tert-butyl ether by a tert-butyl group.

[0055] "Solid-phase synthesis" refers to a synthesis method in which the reactants are attached to an insoluble solid-phase support.

[0056] "Fmoc solid-phase synthesis method" refers to first attaching an N-terminal Fmoc-protected amino acid to a solid-phase support, removing the N-terminal Fmoc protecting group, and then attaching a second N-terminal Fmoc-protected amino acid to the amino acid already attached to the solid-phase support, and repeating the above steps to sequentially attach the remaining amino acids. In the solid-phase synthesis of polypeptides, a resin is usually used as the solid-phase support, and in this application, an amino resin is used as the solid-phase support.

[0057] Some commonly used abbreviations in this application have the following meanings:

[0058] Ac2O: acetic anhydride;

[0059] Boc: tert-butoxycarbonyl;

[0060] CDI: N,N'-carbonyldiimidazole;

[0061] DCM: dichloromethane;

[0062] DIC: N,N'-diisopropylcarbodiimide;

[0063] DIEA: N,N-diisopropylethylamine;

[0064] D-(Diet)Har: D-bisethylhomoarginine;

[0065] (Diet)Har: L-bisethylhomoarginine;

[0066] DMF: N,N-dimethylformamide;

[0067] DODT: 3,6 - dioxaoctane - 1,8 - dithiol;

[0068] EDT: 1,2 - ethanedithiol;

[0069] Fmoc: 9 - fluorenylmethyloxycarbonyl;

[0070] Fmoc - D - Ala - OH: N - (9 - fluorenylmethyloxycarbonyl) - D - alanine;

[0071] Fmoc - D - Cpa - OH: N - (9 - fluorenylmethyloxycarbonyl) - D - 4 - chlorophenylalanine;

[0072] Fmoc - D - Lys(X1) - OH: N - (9 - fluorenylmethyloxycarbonyl) - N'-X1 - D - lysine;

[0073] Fmoc - D - Lys(Mtt) - OH: N - (9 - fluorenylmethyloxycarbonyl) - N'-methyltrityl - D - lysine; Fmoc - D - Nal - OH: N - (9 - fluorenylmethyloxycarbonyl) - 3 - (2 - naphthyl) - D - alanine;

[0074] Fmoc - D - Pal - OH: N - (9 - fluorenylmethyloxycarbonyl) - 3 - (3 - pyridyl) - D - alanine;

[0075] Fmoc - Leu - OH: N - (9 - fluorenylmethyloxycarbonyl) - L - leucine;

[0076] Fmoc - Lys(Mtt) - OH: N - (9 - fluorenylmethyloxycarbonyl) - N'-methyltrityl - L - lysine; Fmoc - Lys(X2) - OH: N - (9 - fluorenylmethyloxycarbonyl) - N'-X2 - L - lysine;

[0077] Fmoc - Pro - OH: N - (9 - fluorenylmethyloxycarbonyl) - L - proline;

[0078] Fmoc - Ser( t Bu) - OH: N - (9 - fluorenylmethyloxycarbonyl) - O - tert - butyl - L - serine;

[0079] Fmoc - Ser(X3) - OH: N - (9 - fluorenylmethyloxycarbonyl) - O - X3 - L - serine;

[0080] Fmoc - Tyr( t Bu) - OH: N - (9 - fluorenylmethyloxycarbonyl) - O - tert - butyl - L - tyrosine;

[0081] Fmoc - Tyr(X4) - OH: N - (9 - fluorenylmethyloxycarbonyl) - O - X4 - L - tyrosine;

[0082] HFIP: hexafluoroisopropanol;

[0083] HOAt: N-Hydroxy-7-azabenzotriazole;

[0084] HOBt: 1-Hydroxybenzotriazole;

[0085] Mmt: Methoxytrityl;

[0086] Mtt: Methyltrityl;

[0087] PIP: Piperidine;

[0088] t Bu: tert-Butyl;

[0089] TES: Triethylsilane;

[0090] TFA: Trifluoroacetic acid;

[0091] TIS: Triisopropylsilane.

[0092] A method for preparing ganirelix according to the present application comprises the following steps:

[0093] (1) Using an amino resin as the starting resin, and sequentially coupling amino acids by the Fmoc solid-phase synthesis method, wherein when coupling the 6th and 8th amino acids at the N-terminus, Fmoc-D-Lys(X1)-OH and Fmoc-Lys(X2)-OH are used respectively, X1 and X2 are the same or different, and are respectively selected from Mtt or Mmt, and when coupling the 4th and 5th amino acids at the N-terminus, side-chain protected Ser and Tyr are used respectively; after coupling is completed, the N-terminal Fmoc protecting group is removed;

[0094] (2) Performing an acetylation reaction on the N-terminus;

[0095] (3) Removing the Mtt and / or Mmt protecting groups on the side chain of Lys;

[0096] (4) Using diethylamine and a catalyst to perform a side-chain modification reaction to convert the 6th and 8th amino acids on the resin obtained in step (3) into D-(Diet)Har and (Diet)Har respectively, and the catalyst is CDI and copper chloride, or triphosgene and dibromotriphenylphosphine;

[0097] (5) Cleaving to obtain ganirelix.

[0098] Ganirelix (SEQ ID NO.1):

[0099] Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-(Diet)Har-Leu-(Diet)Har-Pro-D-Ala-NH2.

[0100] In one embodiment, the synthetic route is as Figure 1 shown.

[0101] The present application will be further described below in conjunction with embodiments; however, these embodiments do not limit the scope of the present application. Unless otherwise stated, all reactants used in each embodiment are obtained commercially; the instruments and equipment used in synthetic experiments and product analysis and detection are all conventional instruments and equipment commonly used in organic synthesis.

[0102] Example 1: Preparation of ganirelix: Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-(Diet)Har-Leu-(Diet)Har-Pro-D-Ala-NH2, and ganirelix acetate

[0103] (1) Preparation of Fmoc-D-Ala-resin complex

[0104] Add 1 g of Rink Amide AM resin (0.5 mmol / g) to a reactor equipped with a filtration device, add DMF to swell for 1 hour, and filter.

[0105] Add a 20% (by volume) PIP / DMF solution to remove the Fmoc protecting group. Detect with ninhydrin. The resin shows a positive result, indicating that the deprotection reaction is complete. Wash the resin 6 times with DMF.

[0106] Take 1.5 mmol of Fmoc-Ala-OH, 1.5 mmol of DIC, and 1.5 mmol of HOBt, dissolve them in 10 mL of DMF, add the resulting solution to the reactor, and react at 30 °C for 1 hour. After the reaction is completed, filter. Then add 10 mL of DMF to wash the resin, wash 6 times and filter. Take a small amount of resin for ninhydrin detection, and the resin shows a negative result.

[0107] (2) Preparation of protected Ac-D-Nal-D-Cpa-D-Pal-Ser( t Bu)-Tyr( t Bu)-D-Lys(Mtt)-Leu-Lys(Mtt)-Pro-D-Ala-NH2-resin complex

[0108] Add 10 mL of a 20% (by volume) PIP / DMF solution to the reactor and react at 30 °C for 20 min. After the reaction is completed, filter. Then add 10 mL of DMF to wash the resin, wash 6 times and filter. Detect with ninhydrin, and the resin is positive.

[0109] Take 1.5 mmol of Fmoc-Pro-OH, 1.5 mmol of DIC, and 1.5 mmol of HOBT, dissolve them in 10 mL of DMF, add the resulting solution to a reactor, and react at 30 °C for 1 hour. After the reaction is completed, filter. Then add 10 mL of DMF to wash the resin, wash 6 times, and filter. Take a small amount of resin for ninhydrin detection, and the resin shows negative.

[0110] Add a 20% (volume ratio) PIP / DMF solution to remove the Fmoc protecting group. React at 30 °C for 20 min. After the reaction is completed, filter. Then add 10 mL of DMF to wash the resin, wash 6 times, and filter. Perform ninhydrin detection, and the resin is positive. Proceed to the next amino acid coupling.

[0111] According to the above coupling method, sequentially couple Fmoc-Lys(Mtt)-OH, Fmoc-Leu-OH, Fmoc-D-Lys(Mtt)-OH, Fmoc-Tyr( t Bu)-OH, Fmoc-Ser( t Bu)-OH, Fmoc-D-Pal-OH, Fmoc-D-Cpa-OH, Fmoc-D-Nal-OH.

[0112] After the coupling is completed, add a 20% (volume ratio) PIP / DMF solution to remove the Fmoc protecting group. React at 30 °C for 20 min. After the reaction is completed, filter. Then add 10 mL of DMF to wash the resin, wash 6 times, and filter. Perform ninhydrin detection, and the resin is positive.

[0113] Take 1.5 mmol of Ac2O and 1.5 mmol of DIEA, dissolve them in 10 mL of DCM, add the resulting solution to a reactor, and react at 30 °C for 1 hour. After the reaction is completed, filter. Then add 10 mL of DMF to wash the resin, wash 6 times, and filter. Take a small amount of resin for ninhydrin detection, and the resin shows negative.

[0114] (3) Prepare the protected Ac-D-Nal-D-Cpa-D-Pal-Ser( t Bu)-Tyr( t Bu)-D-(Diet)Har-Leu-(Diet)Har-Pro-D-Ala-NH2 resin complex.

[0115] Add 10 mL of 30% (by volume) HFIP / DCM to the reactor, react at 30 °C for 30 min, repeat three times, wash with DCM three times, take a small amount of the resin for ninhydrin detection, and the resin shows a positive result. Dissolve 3.0 mmol of diethylamine, 0.75 mmol of CDI, and 0.5 mmol of copper chloride in 10 mL of DCM, add the resulting solution to the reactor, and react at 30 °C for 1 hour. After the reaction is completed, filter. Then add 10 mL of DMF, wash the resin six times, and filter. Take a small amount of the resin for ninhydrin detection, and the resin shows a negative result. Wash with DCM six times and methanol three times, and then dry by suction.

[0116] (4) Prepare the crude product of Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-(Diet)Har-Leu-(Diet)Har-Pro-D-Ala-NH2.

[0117] Add the cleavage reagent (by volume percentage, TFA: EDT: benzyl mercaptan: phenol: water = 90%: 2.5%: 2.5%: 2.5%: 2.5%) to the peptide resin complex obtained in step (3), and react at 0 °C for 2 h. Filter, add the filtrate to 10 times the volume of ether for precipitation, centrifuge to obtain the polypeptide precipitate, and the weight of the crude product is 900 mg.

[0118] (5) Prepare the fine product of Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-(Diet)Har-Leu-(Diet)Har-Pro-D-Ala-NH2.

[0119] Take 900 mg of the crude product (the crude product yield is 114%), perform separation and purification treatment and salt conversion treatment by reverse-phase high-performance liquid chromatography, and then obtain 630 mg (the pure product yield is 80%) of the fine product of ganirelix acetate after freeze-drying treatment. The HPLC and MS spectra of the fine product of ganirelix acetate are as Figures 2-3 shown.

[0120] The packing material of the reverse-phase high-performance liquid chromatography column is C18. The separation and purification treatment conditions and salt conversion treatment conditions are shown in Table 1 and Table 2 respectively.

[0121] Table 1 Separation and purification treatment conditions

[0122] Time / min Phase A (100 mM ammonium acetate aqueous solution, pH = 6.5) / % Phase B (acetonitrile) / % 0 1 99 50 50 50

[0123] Table 2 Salt conversion treatment conditions

[0124] Time / min Phase A (aqueous acetic acid solution with a mass fraction of 0.3%) / % Phase B (acetonitrile) / % 0 1 99 50 50 50

[0125] The HPLC detection conditions for the fine product of ganirelix acetate are as follows:

[0126] Mobile phase: Phase A: 0.065% TFA / Water; Phase B: 0.05% TFA / ACN;

[0127] Column information: C18 (250×4.6 mm, 5 μm);

[0128] Flow rate: 1 mL / min; Wavelength: 220 nm

[0129] The elution gradient is as follows:

[0130] Time (minutes) Phase A (%) Phase B (%) 0 80 20 25 20 80 25.1 5 95 30 5 95 30.1 80 20 35 80 20

[0131] Examples 2 - 6:

[0132] Except for the changes in the conditions listed in the second column of Table 3, the other steps and conditions of Examples 2 - 6 are the same as those of Example 1.

[0133] Table 3 Examples 2 - 6

[0134]

[0135] As can be seen from the examples, in the preparation method of ganirelix in this application, the method of coupling one by one is adopted, using lysine with side-chain protection as the raw material. After N-terminal acetylation, the side-chain protecting group of lysine is removed, and lysine is modified to (Diet)Har on the solid-phase carrier. On the one hand, it avoids the use of amino acid (Diet)Har with high cost and easy decomposition, and on the other hand, the operation is simple, so the process cost is reduced. Diethylamine and a catalyst are used to modify lysine on the solid-phase carrier. Diethylamine and the catalyst used are cheap, and the raw material cost is low; using lysine with Mtt or Mmt protection on the side chain as the raw material, after N-terminal acetylation, the side-chain protecting group can be conveniently and low-costly removed using hexafluoroisopropanol solution. The preparation method of this application has simple operation, low cost, and the yield of ganirelix acetate fine product reaches more than 80%.

[0136] The preferred embodiments of this application have been described in detail above. However, this application is not limited to the specific details in the above embodiments. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solutions of this application, and these simple modifications all belong to the protection scope of this application.

[0137] In addition, it should be noted that, among the various specific technical features described in the above specific embodiments, they can be combined in any suitable way without conflict. To avoid unnecessary repetition, this application will not separately describe various possible combination methods.

[0138] Furthermore, any combination can be made between various different embodiments of this application, as long as it does not violate the idea of this application, it should also be regarded as the content disclosed in this application.

Claims

1. A method for preparing ganirelix, characterized in that: The following steps are involved: (1) Using an amino resin as a starting resin, sequentially coupling amino acids by Fmoc solid phase synthesis method, wherein Fmoc-D-Lys(X1)-OH and Fmoc-Lys(X2)-OH are used for coupling the 6th and 8th amino acids at the N-terminus, respectively, X1 and X2 are the same or different and are selected from Mtt or Mmt, respectively, and N-terminal Fmoc-protected and side chain-protected Ser and Tyr are used for coupling the 4th and 5th amino acids at the N-terminus, respectively; after the coupling is completed, the N-terminal Fmoc protecting group is removed; (2) Acetylation of the N-terminus; (3) removing the Mtt and / or Mmt protecting groups from the Lys side chain; (4) using diethylamine and a catalyst to carry out a side chain modification reaction, converting the 6th and 8th amino acids on the peptide resin complex obtained in step (3) into D-(Diet)Har and (Diet)Har, respectively, wherein the catalyst is CDI and copper chloride, or triphosgene and dibromotriphenylphosphine; (5) Cleavage to obtain ganirelix.

2. The preparation method according to claim 1, characterized in that: In step (4), the molar ratio of diethylamine to the peptide resin complex obtained in step (3) is 2-10:1, preferably 3-6:

1.

3. The preparation method according to claim 1 or 2, characterized in that: In step (4), the molar ratio of the catalyst CDI, copper chloride and the peptide resin complex obtained in step (3) is (1-5): (1-3): 1, preferably (1-3): 1: 1; the molar ratio of the catalyst triphosgene: dibromotriphenylphosphine and the peptide resin complex obtained in step (3) is (1-5): (1-3): 1, preferably (1-3): 1:

1.

4. The preparation method according to claim 2 or 3, characterized in that: In step (4), the reaction temperature of the side chain modification reaction is 20-80°C, preferably 30-60°C; the reaction time is 0.5-3h, preferably 1-2h.

5. The preparation method according to claim 2 or 3, characterized in that: In step (4), the reaction solvent of the side chain modification reaction is selected from one or more of dichloromethane, dichloroethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and dimethyl sulfoxide. Preferably, the reaction solvent is selected from dichloromethane or dichloroethane.

6. The preparation method according to any one of claims 1 to 3, characterized in that: In step (3), a hexafluoroisopropanol solution is used to remove the Mtt and / or Mmt protecting groups; the hexafluoroisopropanol solution is preferably a hexafluoroisopropanol / dichloromethane solution or a hexafluoroisopropanol / dichloroethane solution; more preferably a 20-40% (V) hexafluoroisopropanol / dichloromethane solution or a 20-40% (V) hexafluoroisopropanol / dichloroethane solution.

7. The preparation method according to claim 6, characterized in that: In step (3), the ratio of the hexafluoroisopropanol solution to the peptide resin complex obtained in step (2) is 10-50 mL / mmol resin, preferably 20-30 mL / mmol resin; the reaction temperature of the removal reaction is 20-80°C, preferably 30-60°C, such as 30°C, 40°C, 50°C, 60°C, 70°C, and the reaction time of the removal reaction is 10 min-60 min, preferably 20 min-30 min.

8. The preparation method according to any one of claims 1 to 3, characterized in that: The peptide resin complex obtained in step (3) is Ac-D-Nal-D-Cpa-D-Pal-Ser(X3)-Tyr(X4)-D-Lys-Leu-Lys-Pro-D-Ala-NH-resin; the peptide resin complex obtained in step (4) is Ac-D-Nal-D-Cpa-D-Pal-Ser(X3)-Tyr(X4)-D-(Diet)Har-Leu-(Diet)Har-Pro-D-Ala-NH-resin; X3 and X4 are the same or different and are protecting groups for OH on the side chain; preferably X3 and X4 are selected from t Bu or Boc.

9. The preparation method according to any one of claims 1 to 3, characterized in that: In step (5), ganirelix is ​​obtained by cleavage, precipitation and purification.

10. A method for preparing ganirelix acetate, characterized in that: The method comprises the following steps: obtaining ganirelix according to the preparation method according to any one of claims 1 to 9, and then obtaining ganirelix acetate after salt conversion and optional freeze-drying steps.

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