Preparation method of ganirelix acetate

Abstract

The invention discloses a preparation method of ganirelix acetate, which belongs to the technical field of drug synthesis, and comprises the following preparation steps: synthesizing ganirelix acetate precursor resin I; synthesizing ganirelix precursor resin II; extending a peptide chain: sequentially coupling Fmoc-D-Nal-Ac, Fmoc-D-Phe (4-Cl)-OH, Fmoc-3-(3-Pyridyl)-D-Ala-OH, Fmoc-Ser (tBu)-OH and Fmoc-Tyr (tBu)-OH by taking the ganirelix precursor resin II as a starting point, and removing an Fmoc protecting group after each step of coupling, so as to obtain ganirelix peptide resin; splitting decomposition and purification: splitting decomposition, purification and freeze-drying are performed on the ganirelix peptide resin, and ganirelix acetate is obtained. The progressive synthesis path from the precursor I to the precursor II is clear, the controllability is high, and the method is suitable for large-scale production and has remarkable industrial application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical synthesis technology, and in particular to a method for preparing ganirilac acetate. Background Technology

[0002] Ganirellix Acetate, with the molecular formula C80H113ClN18O13 and a molecular weight of 1570.34, has the CAS registry number 123246-29-7. Its chemical structure is shown below:

[0003]

[0004] Ganirelix acetate is a synthetic decapeptide compound similar to endogenous gonadotropin-releasing hormone (GnRH). It is a GnRH antagonist that competitively antagonizes gonadotropin GnRH receptors, thereby altering the pathway and inducing rapid, reversible, and consistent secretion of gonadotropins. Ganirelix acetate inhibits pituitary LH secretion more significantly than FSH secretion, thus reducing sex hormone production. By inhibiting mid-cycle GnRH-induced LH fluctuations, ganirelix acetate can suppress ovulation, oocyte maturation and division, and luteinization. In women with ovarian hyperstimulation syndrome, ganirelix acetate can prevent LH fluctuations and related stimulation, and improve implantation and pregnancy rates. Therefore, ganirelix acetate has high medicinal value and broad market prospects.

[0005] US4801577, US5212288 and US5767082 disclose a solid-phase synthesis method for ganirielic acetate using the Boc strategy. However, this process generates a lot of waste liquid, is expensive, has a low yield, and causes significant environmental pollution, making it unsuitable for large-scale production.

[0006] CN102584845A discloses a method for preparing ganirilec acetate using the Fmoc strategy. Ganirilec acetate is prepared by solid-phase synthesis using the Fmoc method. However, the preparation, synthesis and purification of Fmoc-homoArg(Et)2-OH and Fmoc-D-homoArg(Et)2-OH are relatively complex, the isomers are not easy to control, the synthesis cycle is long, the cost is high, the yield is low, and there are many impurities, making it unsuitable for industrial production.

[0007] CN104844694A discloses a method for preparing ganirelix acetate, which 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 pre-synthesize ganirelix precursor I, and then modifies the side chain amino groups of Lys and D-Lys in precursor I to obtain ganirelix acetate. CN104231055A discloses a method for preparing ganirelix acetate, which is similar to the former. Both of these patents modify the side chains after the decapeptide synthesis is completed, resulting in large molecular weights, difficult reaction control, and numerous impurities. Therefore, this invention proposes a method for preparing ganirelix acetate. Summary of the Invention

[0008] The purpose of this invention is to provide a solid-phase synthesis method for ganirilac acetate. The technical problem this invention aims to solve is that the synthesis and purification of Fmoc-homoArg(Et)2-OH and Fmoc-D-homoArg(Et)2-OH are complex, isomers are difficult to control, the synthesis cycle is long, the cost is high, the yield is low, and there are many impurities, making them unsuitable for industrial production.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] This invention proposes a method for preparing ganirilac acetate, comprising the following preparation steps:

[0011] S1: Synthetic Ganirex precursor resin I:

[0012] Using an amino resin as a carrier, Fmoc-D-Ala-OH, Fmoc-Pro-OH and Fmoc-Lys(Fmoc)-OH were sequentially coupled. After removing the Fmoc protecting group, it was reacted with an N,N-diethylthiourea derivative to obtain Homo-Arg(Et)2-Pro-D-Ala-amino resin, denoted as Ganirec precursor resin I.

[0013] S2: Synthetic Ganirik precursor resin II:

[0014] Fmoc-Leu-OH and Fmoc-D-Lys(Fmoc)-OH were sequentially coupled onto the Ganirilac precursor resin I. After removing the Fmoc protecting group, it was reacted with an N,N-diethylthiourea derivative to obtain Homo-Arg(Et)2-Leu-Homo-Arg(Et)2-Pro-D-Ala-amino resin, denoted as Ganirilac precursor resin II.

[0015] S3: Peptide chain extension:

[0016] Starting with the ganirilac precursor resin II, Fmoc-D-Nal-Ac, Fmoc-D-Phe(4-Cl)-OH, Fmoc-3-(3-Pyridyl)-D-Ala-OH, Fmoc-Ser(tBu)-OH and Fmoc-Tyr(tBu)-OH are coupled sequentially. After each coupling step, the Fmoc protecting group is removed to obtain the ganirilac peptide resin.

[0017] S4: Pyrolysis and Purification:

[0018] The ganiriplastic peptide resin was cleaved, purified, and freeze-dried to obtain ganiriplastic acetate.

[0019] Preferably, in step S1, the amino resin is selected from Rink Amide resin, Rink Amide-MBHA resin, or Rink Amide-AM resin, and is preferably Rink Amide resin.

[0020] Preferably, the degree of substitution of the amino resin is 0.61-0.81 mmol / g.

[0021] Preferably, in step S3, the coupling agent used in the coupling reaction is selected from the DIC / HOBt, DIEA / HOBt / TBTU, or HATU / HOAt system.

[0022] Preferably, in S1 and S2, the specific steps of the reaction with the N,N-diethylthiourea derivative are as follows:

[0023] First, N,N-diethylthiourea is reacted with hydrogen peroxide in an aqueous solution for 1-3 hours to generate an intermediate. Then, the intermediate is reacted with the side-chain amino groups on the resin at a reaction temperature of 20-30℃.

[0024] Preferably, the molar ratio of N,N-diethylthiourea to hydrogen peroxide is 1:1 to 1:1.5.

[0025] Preferably, the molar ratio of the resin to N,N-diethylthiourea is 1:1.1 to 1:1.5.

[0026] Preferably, the reaction temperature of the hydrogen peroxide and thiourea is controlled below 20°C.

[0027] Preferably, in step S4, the pyrolysis solution used for the pyrolysis is trifluoroacetic acid or a mixture of trifluoroacetic acid containing a scavenging agent, wherein the scavenging agent includes one or more of water, anisole, and 1,2-ethanedithiol.

[0028] Preferably, in step S4, the purification includes adding the lysate dropwise to a low-temperature organic solvent for precipitation and filtration to obtain crude ganirilac peptide, followed by chromatographic purification and lyophilization.

[0029] The present invention has the following technical effects:

[0030] This invention employs a stepwise resin-on-resin derivatization strategy, directly constructing the complex HomoArg(Et)2 structure on the resin. This avoids the challenges of synthesizing and purifying free intermediates, significantly simplifying the process, reducing costs, and improving product purity. The progressive synthetic pathway from precursor I to precursor II is clear, highly controllable, suitable for large-scale production, and has significant industrial application value. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to embodiments, and is intended to illustrate the invention rather than limit it. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications also fall within the protection scope of the present invention.

[0032] The meanings of the abbreviations used in this invention are listed in the table below.

[0033] English abbreviations Chinese meaning DIC N,N'-Diisopropylcarbodiimide HOBt 1-Hydroxybenzotriazole (HOBT) DIEA N,N-Diisopropylethylamine TBTU 2-(1H-benzotriazo-L-1-yl)-1,1,3,3-tetramethylureatetrafluoroborate HATU N,N,N′,N′-Tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate HOAt N-hydroxy-7-azabenzotriazole DMF N,N-Dimethylformamide DCM dichloromethane

[0034] Example 1: Synthesis of Ganirilac acetate:

[0035] 5.5 g of Rink Amide-AM resin with a substitution degree of 0.72 mmol / g was packed into a solid-phase reaction column, washed twice with 40 ml of DMF to swell, and deprotected twice with 32 ml of 20% piperidine / DMF.

[0036] Dissolve 2.6 g of Fmoc-D-Ala-OH and 1.1 g of HOBt in 100 mL of DMF, add 1.24 mL of DIC and activate for 5 minutes. Add the activated solution to a solid-phase reaction column and stir under nitrogen for 2 small-scale tests. Dry the reaction solution, wash once with DMF, and deprotect once with 20% piperidine / DMF.

[0037] Repeat the above steps, using a sequential coupling method, to complete the coupling of Fmoc-Pro-OH and Fmoc-Lys(Fmoc)-OH in turn. After removing Fmoc protection, Lys-Pro-D-Ala-amino resin is obtained.

[0038] Derivatization steps: N,N-diethylthiourea was added to hydrogen peroxide and stirred at room temperature for 2 hours, then Lys-Pro-D-Ala-amino resin was added and stirred for 3 hours. The liquid was removed, and the product was washed 3 times with purified water and 4 times with DMF to obtain Ganirilac precursor resin I.

[0039] Dissolve 2.8 g of Fmoc-Leu-OH and 1.1 g of HOBt in 40 mL of DMF. Activate the solution with 1.24 mL of DIC for 5 minutes under ice bath conditions. Add the activated solution to a solid-phase reaction column and react until the Kaiser test is negative. Dry the reaction solution and deprotect it twice with 20% piperidine / DMF. The Kaiser test is positive after this reaction.

[0040] 4.7 g of Fmoc-D-Lys(Fmoc)-OH and 1.1 g of HOBt were dissolved in 40 mL of DMF. 1.24 mL of DIC was added under ice bath conditions for activation for 5 minutes. The activated solution was then added to a solid-phase reaction column, and the reaction was allowed to proceed until the Kaiser test was negative. The reaction solution was then dried, and deprotected twice with 20% piperidine / DMF. The Kaiser test was positive after this deprotection.

[0041] Derivatization steps: N,N-diethylthiourea was added to hydrogen peroxide and stirred at room temperature for 2 hours. Then Lys-Leu-Lys-Pro-D-Ala-amino resin was added and stirred for 3 hours. The liquid was removed, and the product was washed twice with purified water and twice with DMF to obtain Ganirik precursor resin II.

[0042] Acetate-nirexeptide resin was synthesized by a one-to-one coupling method.

[0043] The pyrolysis steps are as follows: 77 mL of trifluoroacetic acid is measured, cooled to below 20°C, and acetic acid and nirexide resin are slowly added. The temperature is controlled at 20~35°C, and the reaction is carried out for 2 hours. The mixture is filtered, and the filter cake is washed three times with 30 mL of trifluoroacetic acid. The filtrates are then combined and collected.

[0044] The sedimentation and filtration steps are as follows: 60 ml of methyl ether is cooled to 0±10℃, and the filtrate is slowly added while stirring. The reaction temperature is controlled below 40℃. After the addition is complete, stirring is continued for 10 min, and the mixture is allowed to stand for 1 h. The crude product of Ganirik is obtained by filtration with a purity of 91.17%.

[0045] Example 2: Adjusting the resin type:

[0046] Except for replacing the resin with Rink Amide-MBHA resin with a substitution degree of 0.65 mmol / g, the other steps were exactly the same as in Example 1.

[0047] Results: The final purity of ganirielic acetate was 90.85%, and the yield was 76.2%.

[0048] Example 3: Adjusting the resin substitution degree:

[0049] The Rink Amide resin with a substitution degree of 0.81 mmol / g was used, and the remaining steps were the same as in Example 1.

[0050] Results: The final product purity was 91.40%, and the yield was 78.5%.

[0051] Example 4: Adjusting the coupling agent system

[0052] In the coupling reactions in steps (2) and (3), the coupling agent is replaced by the HATU / HOAt / DIEA system (HATU:HOAt:DIEA = 1:1:2), and the remaining steps are the same as in Example 1.

[0053] Results: The final product purity was 91.75%, and the yield was 79.3%.

[0054] Example 5: Adjusting the conditions of the derivatization reaction

[0055] In the derivatization steps for preparing precursor resins I and II, the reaction temperature was controlled at 20°C, the molar ratio of N,N-diethylthiourea to hydrogen peroxide was adjusted to 1:1.2, the resin to thiourea equivalent ratio was 1:1.3, and the reaction time was 2.5 hours. The remaining steps were the same as in Example 1.

[0056] Results: The final product purity was 91.60%, and the yield was 77.8%.

[0057] Comparative Example 1: Synthesis using the traditional Fmoc-HomoArg(Et)2-OH method:

[0058] Following the method disclosed in CN102584845A, solid-phase synthesis was performed directly using Fmoc-HomoArg(Et)2-OH and Fmoc-D-HomoArg(Et)2-OH as raw materials, without employing a resin-based derivatization strategy. The synthesis steps were the same as those for the peptide chain extension in Example 1.

[0059] Results: The final product had a purity of 85.30% and a yield of 62.5%, with a high content of isomer impurities (approximately 3.2%).

[0060] Comparative Example 2: Side chain derivatization after full peptide synthesis:

[0061] Following the method described in CN104844694A, a complete decapeptide resin was first synthesized using Fmoc-Lys(Boc)-OH and Fmoc-D-Lys(Boc)-OH, followed by a side-chain thiourea derivatization reaction. The reaction conditions were the same as in Example 1.

[0062] Results: The final product purity was 88.10%, the yield was 68.7%, the amount of byproducts increased, and purification was difficult.

[0063] Comparative Example 3: Derivatization reaction temperature was too high

[0064] In the derivatization step for preparing precursor resin I, the reaction temperature was increased to 40°C, and the remaining steps were exactly the same as in Example 1.

[0065] Results: The precursor resin I derivatization was incomplete, which hindered the subsequent peptide chain elongation. The final product purity was only 82.50%, the yield was 55.4%, and the color was darker with more impurities.

[0066] Effect Comparison Analysis Table:

[0067] project Final purity (%) Yield (%) Main impurity / isomer content Process controllability Suitable for industrialization level Example 1 91.17 75.5 <1.0% high high Example 2 90.85 76.2 <1.2% high high Example 3 91.40 78.5 <0.9% high high Example 4 91.75 79.3 <0.8% high higher Example 5 91.60 77.8 <1.0% high high Comparative Example 1 85.30 62.5 ≈3.2% Low Low Comparative Example 2 88.10 68.7 ≈2.1% middle middle Comparative Example 3 82.50 55.4 ≈4.5% Low Low

[0068] Conclusion Analysis:

[0069] All embodiments of the present invention exhibit high purity (≥90.85%), high yield (≥75.5%), and low impurity content, indicating that the stepwise resin derivatization strategy has excellent controllability and reproducibility.

[0070] Comparative Example 1 shows that when synthesizing using traditional free HomoArg(Et)2 raw materials, isomer control is difficult, and purity and yield decrease significantly.

[0071] Comparative Example 2 shows that after the synthesis of the whole peptide, the side chain modification results in large steric hindrance, low reaction efficiency, and an increase in byproducts.

[0072] Comparative Example 3 shows that excessively high derivatization reaction temperatures can exacerbate side reactions, severely affecting product quality and yield.

[0073] In summary, the method for preparing ganirilac acetate provided by this invention is significantly superior to existing technologies in terms of process simplicity, product purity, yield, and industrial feasibility, and possesses outstanding technological advancement and practical value.

[0074] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing nirelix acetate, characterized in that, The preparation steps include the following: S1: Synthetic Ganirex precursor resin I: Using an amino resin as a carrier, Fmoc-D-Ala-OH, Fmoc-Pro-OH and Fmoc-Lys(Fmoc)-OH were sequentially coupled. After removing the Fmoc protecting group, it was reacted with an N,N-diethylthiourea derivative to obtain Homo-Arg(Et)2-Pro-D-Ala-amino resin, denoted as Ganirec precursor resin I. S2: Synthetic Ganirik precursor resin II: Fmoc-Leu-OH and Fmoc-D-Lys(Fmoc)-OH were sequentially coupled onto the Ganirilac precursor resin I. After removing the Fmoc protecting group, it was reacted with an N,N-diethylthiourea derivative to obtain Homo-Arg(Et)2-Leu-Homo-Arg(Et)2-Pro-D-Ala-amino resin, denoted as Ganirilac precursor resin II. S3: Peptide chain extension: Starting with the ganirilac precursor resin II, Fmoc-D-Nal-Ac, Fmoc-D-Phe(4-Cl)-OH, Fmoc-3-(3-Pyridyl)-D-Ala-OH, Fmoc-Ser(tBu)-OH and Fmoc-Tyr(tBu)-OH are coupled sequentially. After each coupling step, the Fmoc protecting group is removed to obtain the ganirilac peptide resin. S4: Pyrolysis and Purification: The ganiriplastic peptide resin was cleaved, purified, and freeze-dried to obtain ganiriplastic acetate.

2. The method for preparing ganirilac acetate according to claim 1, characterized in that, In step S1, the amino resin is selected from Rink Amide resin, Rink Amide-MBHA resin, or Rink Amide-AM resin, preferably Rink Amide resin.

3. The method for preparing ganirilac acetate according to claim 1, characterized in that, The degree of substitution of the amino resin is 0.61-0.81 mmol / g.

4. The method for preparing ganirilac acetate according to claim 1, characterized in that, In step S3, the coupling agent used in the coupling reaction is selected from the DIC / HOBt, DIEA / HOBt / TBTU, or HATU / HOAt system.

5. The method for preparing ganirilac acetate according to claim 1, characterized in that, In S1 and S2, the specific steps of the reaction with the N,N-diethylthiourea derivative are as follows: First, N,N-diethylthiourea is reacted with hydrogen peroxide in an aqueous solution for 1-3 hours to generate an intermediate. Then, the intermediate is reacted with the side-chain amino groups on the resin at a reaction temperature of 20-30℃.

6. The method for preparing ganirilac acetate according to claim 5, characterized in that, The molar ratio of N,N-diethylthiourea to hydrogen peroxide is 1:1 to 1:1.

5.

7. The method for preparing ganirilac acetate according to claim 5, characterized in that, The molar ratio of the resin to N,N-diethylthiourea is from 1:1.1 to 1:1.

5.

8. The method for preparing ganirilac acetate according to claim 5, characterized in that, The reaction temperature of hydrogen peroxide and thiourea is controlled below 20°C.

9. The method for preparing ganirilac acetate according to claim 1, characterized in that, In step S4, the pyrolysis solution used is trifluoroacetic acid or a mixture of trifluoroacetic acid containing a scavenging agent, wherein the scavenging agent includes one or more of water, anisole, and 1,2-ethanedithiol.

10. The method for preparing ganirilac acetate according to claim 1, characterized in that, In step S4, the purification includes adding the lysate dropwise to a low-temperature organic solvent for precipitation and filtration to obtain crude ganirilac peptide, which is then purified by chromatography and lyophilized.

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