A method of preparing degarelix

Abstract

The application provides a method for preparing degarelix, introducing a dipeptide fragment Fmoc-Pro-D-Ala-OH, and using a morpholine / DMF deprotecting reagent, so that hydantoin impurities [4Aph(D / L-Hydantoinacetyl) 5 ]Degarelix and [D-Pro 9 ]Deagarelix impurities can be effectively controlled. The application creatively provides a degarelix preparation method which is simple to operate, easy to control in quality, small in environmental pollution and suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to a method for preparing degarelix, belonging to the field of biomedical technology. Background Technology

[0002] Degarelix (brand name Firmagon) is a gonadotropin-releasing hormone (GnRH) receptor inhibitor developed by Ferring GmbH in Denmark and approved by the US FDA on December 24, 2008. Degarelix is ​​primarily used to treat patients with advanced prostate cancer. Its mechanism of action involves inhibiting testosterone, which is crucial for the continued growth of prostate cancer, thereby slowing its growth and progression.

[0003] Degarelk is a linear decapeptide containing seven non-natural amino acids, five of which are D-configured. The decapeptide backbone contains ten chiral centers, and the side chain substituent of the amino acid residue at position 5 contains another chiral center; therefore, degarelk contains a total of eleven chiral centers. The six-membered ring L-Hor of the fifth amino acid, Aph(Hor), in the degarelk structure is highly susceptible to isomerization under basic conditions, generating the 5-hydantoin-5-acetylated impurity 4Aph(D / L-Hydantoinacetyl). 5 Degarelix. Additionally, the 9th amino acid in degarelix is ​​proline, making it extremely easy to generate racemic proline impurities [D-Pro] during stepwise coupling. 9 Degarelix.

[0004] WO0984663 is the earliest synthetic patent, reporting a solid-phase synthesis method using Boc-protected amino acids by degarelek. The 4-aminophenylalanine (Aph) at positions 5 and 6 requires protection with an Fmoc protecting group, followed by removal with a 25% piperidine / DMF solution. This is then reacted with L-hydrogenated orotic acid or tert-butyl isocyanate, and the next amino acid is sequentially linked. Finally, after acetic anhydride acylation, the protecting group and cleaved peptide are removed with HF. Another method is mentioned where orotic acid is introduced onto the side chain of the 5-amino acid after the entire peptide chain is complete, employing a Boc solid-phase synthesis strategy. The removal of the temporary protecting group in this method is done under acidic conditions (TFA). While this can partially avoid the isomerization of Hor in the 5-amino acid Aph (Hor) to generate hydantoin-5-acetic acid, the Boc strategy ultimately requires HF cleavage, which poses a significant hazard to humans and the environment.

[0005] Patents CN102952174 and CN102329373 describe a solid-phase synthesis method using Fmoc-protected amino acids as starting materials. DIC / HOBt is used as a coupling agent for peptide linking. The hydrogenated orotic acid fragment linked to the amino group at the para-position of the 5-position phenylalanine ring is first protected with triphenyl and then introduced last, avoiding rearrangement side reactions. D-4Aph(Cbm) is used instead of D-4Aph(Cbm-tBu) to avoid the difficulty of t-Bu removal and reduce side reactions. However, introducing the hydrogenated orotic acid at the para-position of the 5-position phenylalanine ring after the entire peptide chain is complete results in an unstable process for selectively removing Trt, making it unsuitable for industrial production.

[0006] Patent CN103351428 discloses a method for solid-phase fragment synthesis of degarelk, employing a fragment 6+4 strategy. This involves selectively removing the X residue from the 6th amino acid residue, attaching L-Hor to its side-chain amino group, removing Fmoc, and then solid-phase coupling with an acetyl-terminated acidic fragment 4. The resulting fragment is then cleaved and purified to obtain the target product. This route is extremely cumbersome and unsuitable for industrial production.

[0007] Patent CN102428097 describes a solid-phase synthesis strategy using Fmoc with 20% piperidine / DMF as the deprotection solution, gradually coupling the corresponding amino acids. Although the hydantoin impurity generated by the rearrangement of the 5-position amino acid in 20% piperidine / DMF can be controlled to below 0.3% by weight, it cannot effectively avoid the racemic impurity [D-Pro] of Pro. 9 The formation of Degarelix. The specific structure of the impurity is as follows:

[0008] Summary of the Invention

[0009] This invention provides a method for preparing degarelk, overcoming the shortcomings of the prior art.

[0010] A method for preparing degarelk includes the following steps: using Rink amino resin as the starting resin, starting from an Fmoc-protected amino acid, wherein a dipeptide fragment Fmoc-Pro-D-Ala-OH is introduced between the 9th and 10th positions, solid-phase synthesis is adopted, and a peptide-linking reaction is carried out with a coupling agent to sequentially link the Fmoc-protected amino acid to obtain a protected decapeptide resin, a deprotecting agent is used to sequentially remove the Fmoc group from the amino acid, a cleavage reagent is used for cleavage, and after purification, the degarelk product is obtained.

[0011] The deprotection agent is selected from piperidine / DMF and morpholine / DMF, with morpholine / DMF being preferred.

[0012] As described above, a method for preparing degarelk specifically includes the following steps:

[0013] 1) Using Fmoc-protected Rink amino resin as the starting resin, the dipeptide fragment Fmoc-Pro-D-Ala-OH was introduced, and morpholine / DMF was used as the deprotection agent to couple it with Rink resin to obtain Fmoc-Pro-D-Ala-Rink resin.

[0014] 2) Using the Fmoc solid-phase synthesis method, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph(Cbm)-OH, Fmoc-4Aph(Hor)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-4Cpa-OH and Fmoc-D-2Nal-OH were sequentially coupled with coupling agents according to the amino acid sequence of degarelcke, with morpholine / DMF used as the deprotection agent during the process;

[0015] 3) Morpholine / DMF was used as the deprotection agent to remove the N-terminal Fmoc of degarelcki, followed by acetyl capping, washing, and drying to obtain peptide resin;

[0016] 4) Cut the peptide resin with a cleavage reagent, precipitate, and centrifuge to obtain crude peptide;

[0017] 5) The crude peptide was purified to obtain Degarelek peptide.

[0018] In the above method for preparing degarelic, the amino resin mentioned in step 1) is selected from one of Rink amide AM and Rink amide MBHA;

[0019] In the above-mentioned method for preparing degarelic, the degree of substitution of the amino resin in step 1) is 0.4 to 0.9 mmol / g.

[0020] The dipeptide Fmoc-Pro-D-Ala-OH can be synthesized by solid-phase or liquid-phase synthesis. Preferably, the dipeptide Fmoc-Pro-D-Ala-OH is synthesized by liquid-phase synthesis.

[0021] The coupling agent is selected from carbodiimide type compounds or onium salt type compounds.

[0022] The coupling agent is selected from DIC / HOBt, EDCI / HOBt, HBTU / DIEA / HOBt, TBTU / DIEA / HOBt, wherein the preferred coupling agent is DIC / HOBt.

[0023] The deprotecting agent morpholine / DMF has a volume ratio concentration of 20% to 50%, preferably 20%.

[0024] The lysis reagent is selected from TFA / TIS / H2O, TFA / TIS, and TFA / H2O, with TFA / TIS / H2O being the preferred lysis reagent.

[0025] The solid-phase synthesis of the dipeptide Fmoc-Pro-D-Ala-OH specifically involves using CTC resin or Wang resin as the starting resin, gradually coupling Fmoc-D-Ala-OH and Fmoc-Pro-OH, followed by cleavage and recrystallization to obtain Fmoc-Pro-D-Ala-OH.

[0026] The liquid-phase synthesis of the dipeptide Fmoc-Pro-D-Ala-OH specifically involves the formation of the active ester Fmoc-D-Ala-OSu under DCC / HOSu conditions, followed by reaction with H-Pro-OH in Na2CO3 under THF / H2O solvent to generate the dipeptide Fmoc-Pro-D-Ala-OH. Recrystallization from isopropyl ether yields the final dipeptide product. Alternatively, using CTC resin or Wang resin as the starting resin, Fmoc-D-Ala-OH and Fmoc-Pro-OH are progressively coupled, cleaved, and recrystallized to obtain Fmoc-Pro-D-Ala-OH.

[0027] In the above-mentioned degarelic preparation method, the acetyl-capping method in step 3) involves using acetic anhydride and DIEA in molar amounts of 1.5 to 2 times the N-terminal free amino group.

[0028] In the above-mentioned degarelic preparation method, the acetyl capping in step 3) is carried out at 0-5°C.

[0029] In the above-mentioned degarelk preparation method, the washing method in step 3) is to wash with DMF and DCM sequentially.

[0030] In the above-mentioned method for preparing degarelk, the separation and purification in step 5 includes the following steps: the crude peptide is dissolved in water, acetonitrile and acetic acid, filtered through a filter membrane, purified in one step using a TEAP / acetonitrile system, then converted to salt using an ammonium acetate / acetonitrile and acetic acid / acetonitrile system, concentrated and lyophilized.

[0031] Compared with existing technologies, the advantages of this invention are as follows: This invention provides a method for preparing degarelic acid that is simple to operate, easy to control in quality, has low environmental pollution, and is suitable for large-scale production. Using morpholine / DMF as the deprotection agent can effectively control hydantoin impurities [4Aph(D / L-Hydantoinacetyl]]. 5 Degarelix; the introduction of the dipeptide fragment Fmoc-Pro-D-Ala-OH can effectively control [D-Pro] 9 Degarelix impurities. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to further understand the present invention. These embodiments should not be construed as limiting the scope of protection.

[0033] The Chinese names corresponding to the English abbreviations involved in this invention are shown in Table 1:

[0034] Table 1 shows the Chinese names corresponding to the English abbreviations involved in this invention.

[0035]

[0036] The technical solution of the present invention will be further described in detail according to the following embodiments.

[0037] Example 1: Synthesis of Fmoc-Pro-D-Ala-Rink MBHA Resin

[0038] Weigh 75 g (30 mmol, substitution degree: 0.40 mmol / g) of Rink Amide MBHA resin into a solid-phase reaction column. Add 500 mL of DMF to swell for 30 min, then remove the DMF. Wash the resin with 3 × 400 mL of DMF, then remove the DMF. Add 400 mL of DBLK solution (20% morpholine / DMF solution, v / v) for deprotection twice, the first time for 5 min and the second time for 15 min. After deprotection, wash the resin with 400 mL of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod; a positive result for ninhydrin testing indicates that Fmoc has been deprotected.

[0039] Weigh 40.24 g of Fmoc-Pro-D-Ala-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2 h. If the ninhydrin test is negative, remove the reaction solution, add 400 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution to obtain Fmoc-Pro-D-Ala-Rink MBHA resin.

[0040] Example 2: Synthesis of Degarelkeptide Resin

[0041] Weigh 30 mmol of the Fmoc-Pro-D-Ala-Rink MBHA resin obtained in Example 1 into a solid-phase reaction column. Wash the resin with 3 × 400 mL of DMF and remove the DMF. Add 400 mL of DBLK solution (20% morpholine / DMF solution, v / v) for deprotection twice, the first time for 5 min and the second time for 15 min. After deprotection, wash the resin with 400 mL of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. The Chloranil test is positive, indicating that Fmoc has been deprotected.

[0042] Weigh 30.64 g of Fmoc-ILys(Boc)-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve, and after complete dissolution, cool the solution to below 5°C. Then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2 h. After a negative Chloranil test, remove the reaction solution, add 400 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution and continue with the coupling of the next amino acid.

[0043] Add 400 ml of DBLK solution (20% morpholine / DMF solution, v / v) and deprotect twice, the first time for 5 min and the second time for 15 min. After deprotection, wash the resin with 400 ml of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. The ninhydrin test is positive, indicating that Fmoc has been deprotected.

[0044] Weigh 21.21 g of Fmoc-Leu-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2 h. If the ninhydrin test is negative, remove the reaction solution, add 400 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution and continue with the coupling of the next amino acid.

[0045] Following the Fmoc-Leu-OH coupling procedure, Fmoc-D-4Aph(Cbm)-OH (26.73g), Fmoc-4Aph(Hor)-OH (32.55g), Fmoc-Ser(tBu)-OH (23.01g), Fmoc-D-3Pal-OH (23.32g), Fmoc-D-4Cpa-OH (25.31g), and Fmoc-D-2Nal-OH (26.51g) were sequentially linked according to the degarelac amino acid sequence.

[0046] Add 400 ml of DBLK solution (20% morpholine / DMF solution, v / v) and deprotect twice, the first time for 5 min and the second time for 15 min. After deprotection, wash the resin with 400 ml of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. If the ninhydrin test is positive, it indicates that Fmoc has been deprotected.

[0047] Weigh 4.59g Ac₂O (1.5eq) and 5.82g DIEA (1.5eq), add 360ml DMF, mix well, and cool the solution to below 5℃ before pouring it into the reaction column. After reacting at 0-5℃ for 20 min, the ninhydrin test was negative. The reaction solution was then removed. The resin was washed with 400ml DMF each time, 6 times, followed by 400ml DCM each time, 4 times. Finally, the resin was soaked in 400ml DCM for 20 min, and the DCM was removed. The resin was vacuum dried at 20-30℃ until it became a quicksand-like consistency. The dried peptide resin weighed 117.83g, with a resin weight gain of 97%.

[0048] Example 3: Cleavage of degarelicin resin

[0049] Take 1100 ml of the frozen lysis buffer (not higher than 10℃), prepare it with a ratio of TFA:TIS::H2O = 92.5:2.5:2.5 (v / v / v), add 110 g (28 mmol) of the degarelic peptide resin obtained in Example 2, react at 20-30℃ for 2.0 h, filter, wash the resin with TFA, concentrate the filtrate to 1 / 2-2 / 3 volume, then add 10 times the volume of isopropyl ether for precipitation, centrifuge to collect the precipitate, wash 6 times with isopropyl ether, and vacuum dry to obtain 41.38 g of crude peptide, with a yield of 98.3%.

[0050] Example 4: Purification of crude degarelix peptide

[0051] The crude degarelic peptide obtained in Example 3 was dissolved in 20% acetonitrile / water and acetic acid. It was first purified in one step using a 0.5% TEAP (pH 2.25) / acetonitrile system. The fraction with a purity greater than 98% was collected for a two-step salt conversion using ammonium acetate. Elution was performed using a 1.0% acetic acid / acetonitrile system, and the product was concentrated and lyophilized to obtain 19.33 g, a yield of 42.3%. The purity was 99.7%, and the impurity was [4Aph(D / L-Hydantoinacetyl)]. 5 Degarelix not detected, impurity [D-Pro] 9 Degarelix's value was 0.04%.

[0052] Example 5: Synthesis of Fmoc-Pro-D-Ala-Rink MBHA Resin

[0053] Weigh 43.5 g (30 mmol, degree of substitution: 0.69 mmol / g) of Rink Amide MBHA resin into a solid-phase reaction column. Add 250 mL of DMF to swell for 30 min, then remove the DMF. Wash the resin with 3 × 200 mL of DMF, then remove the DMF. Add 200 mL of DBLK solution (35% morpholine / DMF solution, v / v) for deprotection twice, the first time for 15 min and the second time for 30 min. After deprotection, wash the resin with 200 mL of DMF each time, washing 6 times. After the fourth wash, pick up a small amount of resin with a glass rod; a positive result for ninhydrin testing indicates that Fmoc has been deprotected.

[0054] Weigh 40.24 g of Fmoc-Pro-D-Ala-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2–3 h. When the ninhydrin test is negative, remove the reaction solution, add 200 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution to obtain Fmoc-Pro-D-Ala-Rink MBHA resin.

[0055] Example 6: Synthesis of Degarelkeptide Resin

[0056] Weigh 30 mmol of the Fmoc-Pro-D-Ala-Rink MBHA resin obtained in Example 5 into a solid-phase reaction column. Wash the resin with 3 × 200 ml DMF and remove the DMF. Add 200 ml of DBLK solution (35% morpholine / DMF solution, v / v) and deprotect twice, the first time for 15 min and the second time for 30 min. After deprotection, wash the resin with 200 ml of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. The Chloranil test is positive, indicating that Fmoc has been deprotected.

[0057] Weigh 30.64 g of Fmoc-ILys(Boc)-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2 h. When the Chloranil test is negative, remove the reaction solution, add 200 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution and continue with the coupling of the next amino acid.

[0058] Add 200 ml of DBLK solution (35% morpholine / DMF solution, v / v) and deprotect twice, the first time for 15 min and the second time for 30 min. After deprotection, wash the resin with 200 ml of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. If the ninhydrin test is positive, it indicates that Fmoc has been deprotected.

[0059] Weigh 21.21 g of Fmoc-Leu-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2 h. If the ninhydrin test is negative, remove the reaction solution, add 200 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution and continue with the coupling of the next amino acid.

[0060] Following the Fmoc-Leu-OH coupling procedure, Fmoc-D-4Aph(Cbm)-OH (26.73g), Fmoc-4Aph(Hor)-OH (32.55g), Fmoc-Ser(tBu)-OH (23.01g), Fmoc-D-3Pal-OH (23.32g), Fmoc-D-4Cpa-OH (25.31g), and Fmoc-D-2Nal-OH (26.51g) were sequentially linked according to the degarelac amino acid sequence.

[0061] Add 200 ml of DBLK solution (30% morpholine / DMF solution, v / v) and deprotect twice, the first time for 15 min and the second time for 30 min. After deprotection, wash the resin with 200 ml of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. The ninhydrin test is positive, indicating that Fmoc has been deprotected.

[0062] Weigh 5.51g Ac₂O (1.8 eq) and 6.98g DIEA (1.8 eq), add 360ml DMF, mix well, and cool the solution to below 5℃ before pouring it into the reaction column. After reacting at 0–5℃ for 20 min, the ninhydrin test was negative. The reaction solution was then removed. The resin was washed with 200ml DMF each time, 6 times, followed by 4 washes with 200ml DCM each time. Finally, the resin was soaked in 200ml DCM for 20 min, and the DCM was removed. The resin was vacuum dried at 20–30℃ until it became a quicksand-like consistency. The weight of the dried peptide resin was 86.38g, with a resin weight gain of 96%.

[0063] Example 7: Cleavage of degarelckidet peptide resin

[0064] Take 800 ml of the frozen lysis buffer (not exceeding 10°C), prepare it with a ratio of TFA:TIS::H2O = 92.5:2.5:2.5 (V / V / V), add 80 g (28 mmol) of the degarelic peptide resin obtained in Example 6, react at 20-30°C for 2.0 h, filter, wash the resin with TFA, concentrate the filtrate to 1 / 2-2 / 3 volume, then add 10 times the volume of isopropyl ether for precipitation, centrifuge to collect the precipitate, wash 6 times with isopropyl ether, and vacuum dry to obtain 40.41 g of crude peptide, with a yield of 97.8%.

[0065] Example 8: Purification of crude degarelcke peptide

[0066] The crude degarelic peptide obtained in Example 7 was dissolved in 20% acetonitrile / water and acetic acid. It was first purified in one step using a 0.5% TEAP (pH 2.25) / acetonitrile system. The fraction with a purity greater than 98% was collected for a two-step salt conversion using ammonium acetate, followed by elution with a 1.0% acetic acid / acetonitrile system. The purified peptide was concentrated and lyophilized to obtain 18.95 g, with a yield of 41.8%. The purity was 99.5%, and the impurity [4Aph(D / L-Hydantoinacetyl)] was absent. 5 Degarelix not detected %, impurities [D-Pro] 9 Deagarelix's level is 0.07%.

[0067] Example 9: Synthesis of Fmoc-Pro-D-Ala-Rink MBHA Resin

[0068] Weigh 33.3 g (30 mmol, substitution degree: 0.90 mmol / g) of Rink Amide MBHAresin into a solid-phase reaction column. Add 200 mL of DMF to swell for 30 min, then remove the DMF. Wash the resin with 3 × 200 mL of DMF, then remove the DMF. Add 200 mL of DBLK solution (50% morpholine / DMF solution, v / v) for deprotection twice, the first time for 30 min, the second time for 30 min. After each deprotection, wash the resin with 200 mL of DMF, washing 6 times. After the fourth wash, pick up a small amount of resin with a glass rod; a positive result for ninhydrin testing indicates that Fmoc has been deprotected.

[0069] Weigh 40.24 g of Fmoc-Pro-D-Ala-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 3–5 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2–3 h. When the ninhydrin test is negative, remove the reaction solution, add 200 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution to obtain Fmoc-Pro-D-Ala-RinkMBHA resin.

[0070] Example 10 Synthesis of Degarelkeptide Resin

[0071] Weigh 30 mmol of the Fmoc-Pro-D-Ala-Rink MBHA resin obtained in Example 9 into a solid-phase reaction column. Wash the resin with 3 × 200 ml DMF and remove the DMF. Add 200 ml of DBLK solution (50% morpholine / DMF solution, v / v) and deprotect twice, the first time for 30 min and the second time for 30 min. After deprotection, wash the resin with 200 ml DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. The Chloranil test is positive, indicating that Fmoc has been deprotected.

[0072] Weigh 30.64 g of Fmoc-ILys(Boc)-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2 h. When the Chloranil test is negative, remove the reaction solution, add 200 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution and continue with the coupling of the next amino acid.

[0073] Add 200 ml of DBLK solution (50% morpholine / DMF solution, v / v) and deprotect twice, the first time for 30 min and the second time for 30 min. After deprotection, wash the resin with 200 ml of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. If the ninhydrin test is positive, it indicates that Fmoc has been deprotected.

[0074] Weigh 21.21 g of Fmoc-Leu-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2 h. If the ninhydrin test is negative, remove the reaction solution, add 200 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution and continue with the coupling of the next amino acid.

[0075] Following the Fmoc-Leu-OH coupling procedure, Fmoc-D-4Aph(Cbm)-OH (26.73g), Fmoc-4Aph(Hor)-OH (32.55g), Fmoc-Ser(tBu)-OH (23.01g), Fmoc-D-3Pal-OH (23.32g), Fmoc-D-4Cpa-OH (25.31g), and Fmoc-D-2Nal-OH (26.51g) were sequentially linked according to the degarelac amino acid sequence.

[0076] Add 200 ml of DBLK solution (50% morpholine / DMF solution, v / v) and deprotect twice, the first time for 30 min and the second time for 30 min. After deprotection, wash the resin with 200 ml of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. The ninhydrin test is positive, indicating that Fmoc has been deprotected.

[0077] Weigh 6.13g Ac₂O (2.0 eq) and 7.75g DIEA (2.0 eq), add 360ml DMF, mix well, and cool the solution to below 5℃ before pouring it into the reaction column. After reacting at 0–5℃ for 20 min, the ninhydrin test was negative. The reaction solution was then removed. The resin was washed 6 times with 200ml DMF each time, followed by 4 times with 200ml DCM each time. Finally, the resin was soaked in 200ml DCM for 20 min, and the DCM was removed. The resin was vacuum dried at 20–30℃ until it became a quicksand-like consistency. The dried peptide resin weighed 75.82g, with a resin weight gain of 95%.

[0078] Example 11: Cleavage of degarelcki peptide resin

[0079] Take 700 ml of the frozen lysis buffer (not exceeding 10°C), prepare it with a ratio of TFA:TIS::H2O = 92.5:2.5:2.5 (V / V / V), add 70 g (28 mmol) of the degarelic peptide resin obtained in Example 10, react at 20-30°C for 2.0 h, filter, wash the resin with TFA, concentrate the filtrate to 1 / 2-2 / 3 volume, then add 10 times the volume of isopropyl ether for precipitation, centrifuge to collect the precipitate, wash 6 times with isopropyl ether, and vacuum dry to obtain 38.81 g of crude peptide, with a yield of 95.2%.

[0080] Example 12 Purification of crude degarelcke peptide

[0081] The crude degarelic peptide obtained in Example 11 was dissolved in 20% acetonitrile / water and acetic acid. It was first purified in one step using a 0.5% TEAP (pH 2.25) / acetonitrile system. The fraction with a purity greater than 98% was collected for a two-step salt conversion using ammonium acetate. Elution was performed using a 1.0% acetic acid / acetonitrile system, followed by concentration and lyophilization to obtain 18.39 g, yield 40.7%, purity 99.4%, and no impurities [4Aph(D / L-Hydantoinacetyl)]. 5 Degarelix is ​​0.05%, impurities [D-Pro] 9 Deagarelix's concentration is 0.06%.

[0082] Example 13 Synthesis of Fmoc-Pro-D-Ala-Rink AM Resin

[0083] Weigh 38.5 g (30 mmol, degree of substitution: 0.78 mmol / g) of Rink Amide AM resin into a solid-phase reaction column. Add 200 mL of DMF to swell for 30 min, then remove the DMF. Wash the resin with 3 × 200 mL of DMF, then remove the DMF. Add 200 mL of DBLK solution (20% morpholine / DMF solution, v / v) for deprotection twice, the first time for 5 min and the second time for 15 min. After deprotection, wash the resin with 200 mL of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod; the ninhydrin test is positive, indicating that Fmoc has been deprotected.

[0084] Weigh 40.24 g of Fmoc-Pro-D-Ala-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2 h. After a negative ninhydrin test, remove the reaction solution, add 200 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution to obtain Fmoc-Pro-D-Ala-Rink MBHA resin.

[0085] Example 14 Synthesis of Degarelkeptide Resin

[0086] Weigh 30 mmol of the Fmoc-Pro-D-Ala-Rink AM resin obtained in Example 13 into a solid-phase reaction column. Wash the resin with 3 × 200 ml DMF and remove the DMF. Add 200 ml of DBLK solution (20% morpholine / DMF solution, v / v) and deprotect twice, the first time for 5 min and the second time for 15 min. After deprotection, wash the resin with 200 ml DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. The Chloranil test is positive, indicating that Fmoc has been deprotected.

[0087] Weigh 30.64 g of Fmoc-ILys(Boc)-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2 h. When the Chloranil test is negative, remove the reaction solution, add 200 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution and continue with the coupling of the next amino acid.

[0088] Add 200 ml of DBLK solution (20% morpholine / DMF solution, v / v) and deprotect twice, the first time for 5 min and the second time for 15 min. After deprotection, wash the resin with 200 ml of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. The ninhydrin test is positive, indicating that Fmoc has been deprotected.

[0089] Weigh 21.21 g of Fmoc-Leu-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2 h. If the ninhydrin test is negative, remove the reaction solution, add 200 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution and continue with the coupling of the next amino acid.

[0090] Following the Fmoc-Leu-OH coupling procedure, Fmoc-D-4Aph(Cbm)-OH (26.73g), Fmoc-4Aph(Hor)-OH (32.55g), Fmoc-Ser(tBu)-OH (23.01g), Fmoc-D-3Pal-OH (23.32g), Fmoc-D-4Cpa-OH (25.31g), and Fmoc-D-2Nal-OH (26.51g) were sequentially linked according to the degarelac amino acid sequence.

[0091] Add 200 ml of DBLK solution (20% morpholine / DMF solution, v / v) and deprotect twice, the first time for 5 min and the second time for 15 min. After deprotection, wash the resin with 200 ml of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. If the ninhydrin test is positive, it indicates that Fmoc has been deprotected.

[0092] Weigh 4.59g Ac₂O (1.5eq) and 5.82g DIEA (1.5eq), add 360ml DMF, mix well, and cool the solution to below 5℃ before pouring it into the reaction column. After reacting at 0-5℃ for 20 min, the ninhydrin test was negative. The reaction solution was then removed. The resin was washed with 200ml DMF each time, 6 times, followed by 4 washes with 200ml DCM each time. Finally, the resin was soaked in 200ml DCM for 20 min, and the DCM was removed. The resin was vacuum dried at 20-30℃ until it became a quicksand-like consistency. The dried peptide resin weighed 114.83g, with a resin weight gain of 94%.

[0093] Example 15: Cleavage of degarelcki peptide resin

[0094] Take 1100 ml of the frozen lysis buffer (not exceeding 10°C), prepare it with a ratio of TFA:TIS::H2O = 92.5:2.5:2.5 (V / V / V), add 110 g (28.7 mmol) of the degarelic peptide resin obtained in Example 14, react at 20-30°C for 2.0 h, filter, wash the resin with TFA, concentrate the filtrate to 1 / 2-2 / 3 volume, then add 10 times the volume of isopropyl ether for precipitation, collect the precipitate by centrifugation, wash 6 times with isopropyl ether, and vacuum dry to obtain 46.71 g of crude peptide, with a yield of 99.7%.

[0095] Example 16 Purification of crude degarelcke peptide

[0096] The crude degarelic peptide obtained in Example 15 was dissolved in 20% acetonitrile / water and acetic acid. It was first purified in one step using a 0.5% TEAP (pH 2.25) / acetonitrile system. The fraction with a purity greater than 98% was collected for a two-step salt conversion using ammonium acetate. Elution was performed using a 1.0% acetic acid / acetonitrile system, and the product was concentrated and lyophilized to obtain 19.63 g, a yield of 41.9%. The purity was 99.4%, and the impurity was [4Aph(D / L-Hydantoinacetyl)]. 5 Degarelix not detected, impurity [D-Pro] 9 Degarelix is ​​0.05%.

[0097] Example 17 Synthesis of Fmoc-Pro-D-Ala-Rink AM Resin

[0098] Weigh 38.5 g (30 mmol, degree of substitution: 0.78 mmol / g) of Rink Amide AM resin into a solid-phase reaction column. Add 200 mL of DMF to swell for 30 min, then remove the DMF. Wash the resin with 3 × 200 mL of DMF, then remove the DMF. Add 200 mL of DBLK solution (20% piperidine / DMF solution, v / v) for deprotection twice, the first time for 5 min and the second time for 15 min. After deprotection, wash the resin with 200 mL of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod; the ninhydrin test is positive, indicating that Fmoc has been deprotected.

[0099] Weigh 40.24 g of Fmoc-Pro-D-Ala-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2 h. After a negative ninhydrin test, remove the reaction solution, add 200 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution to obtain Fmoc-Pro-D-Ala-Rink MBHA resin.

[0100] Example 18 Synthesis of Degarelkeptide Resin

[0101] Weigh 30 mmol of the Fmoc-Pro-D-Ala-Rink MBHA resin obtained in Example 17 into a solid-phase reaction column. Wash the resin with 3 × 200 mL of DMF and remove the DMF. Add 200 mL of DBLK solution (20% piperidine / DMF solution, v / v) and deprotect twice, the first time for 5 min and the second time for 15 min. After deprotection, wash the resin with 200 mL of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. The Chloranil test is positive, indicating that Fmoc has been deprotected.

[0102] Weigh 30.64 g of Fmoc-ILys(Boc)-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2 h. When the Chloranil test is negative, remove the reaction solution, add 200 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution and continue with the coupling of the next amino acid.

[0103] Add 200 ml of DBLK solution (20% piperidine / DMF solution, v / v) and deprotect twice, the first time for 5 min and the second time for 15 min. After deprotection, wash the resin with 200 ml of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. If the ninhydrin test is positive, it indicates that Fmoc has been deprotected.

[0104] Weigh 21.21 g of Fmoc-Leu-OH and 9.73 g of HOBt, add 120 ml of DMF to dissolve. After complete dissolution, cool the solution to below 5°C, then add 11.36 g of DIC (pre-cooled to <0°C) and activate in the solution for about 4 min. Add the activated solution to the reaction column in a controlled manner and react at 20–30°C for 2 h. If the ninhydrin test is negative, remove the reaction solution, add 200 ml of DMF to wash the resin, and wash 6 times. After washing, remove the washing solution and continue with the coupling of the next amino acid.

[0105] Following the Fmoc-Leu-OH coupling procedure, Fmoc-D-4Aph(Cbm)-OH (26.73g), Fmoc-4Aph(Hor)-OH (32.55g), Fmoc-Ser(tBu)-OH (23.01g), Fmoc-D-3Pal-OH (23.32g), Fmoc-D-4Cpa-OH (25.31g), and Fmoc-D-2Nal-OH (26.51g) were sequentially linked according to the degarelac amino acid sequence.

[0106] Add 200 ml of DBLK solution (20% piperidine / DMF solution, v / v) and deprotect twice, the first time for 5 min and the second time for 15 min. After deprotection, wash the resin with 200 ml of DMF each time, washing 6 times. After the 4th wash, pick up a small amount of resin with a glass rod. If the ninhydrin test is positive, it indicates that Fmoc has been deprotected.

[0107] Weigh 4.59g Ac₂O (1.5eq) and 5.82g DIEA (1.5eq), add 360ml DMF, mix well, and cool the solution to below 5℃ before pouring it into the reaction column. After reacting at 0-5℃ for 20 min, the ninhydrin test was negative. The reaction solution was then removed. The resin was washed with 200ml DMF each time, 6 times, followed by 4 washes with 200ml DCM each time. Finally, the resin was soaked in 200ml DCM for 20 min, and the DCM was removed. The resin was vacuum dried at 20-30℃ until it became a quicksand-like consistency. The dried peptide resin weighed 113.24g, with a resin weight gain of 93%.

[0108] Example 19: Cleavage of degarelcki peptide resin

[0109] Take 1100 ml of the frozen lysis buffer (not higher than 10°C), prepare it with a ratio of TFA:TIS:H2O = 92.5:2.5:2.5 (V / V / V), add 110 g (29.1 mmol) of the degarelic peptide resin obtained in Example 18, react at 20-30°C for 2.0 h, filter, wash the resin with TFA, concentrate the filtrate to 1 / 2-2 / 3 volume, then add 10 times the volume of isopropyl ether for precipitation, centrifuge to collect the precipitate, wash 6 times with isopropyl ether, and vacuum dry to obtain 47.46 g of crude peptide, with a yield of 99.8%.

[0110] Example 20: Purification of crude degarelcke peptide

[0111] The crude degarelic peptide obtained in Example 19 was dissolved in 20% acetonitrile / water and acetic acid. It was first purified in one step using a 0.5% TEAP (pH 2.25) / acetonitrile system. The fraction with a purity greater than 98% was collected for a two-step salt conversion using ammonium acetate. Elution was performed using a 1.0% acetic acid / acetonitrile system, and the product was concentrated and lyophilized to obtain 18.19 g, a yield of 39.8%. The purity was 99.3%, and the impurity [4Aph(D / L-Hydantoinacetyl)] was absent. 5 Degarelix was 0.08%, and impurities [D-Pro] were present. 9 Degarelix was 0.07%.

[0112] Example 21 Synthesis of Fmoc-Pro-D-Ala-OH

[0113] Weigh 30 g (30 mmol, 1.0 mmol / g) of Wang Resin and add it to a solid-phase synthesis column. Wash once with 200 ml of DMF, dry under vacuum, add another 200 ml of DMF to swell for 30 min, and dry under vacuum. Weigh 18.68 g of Fmoc-D-Ala-OH and 9.73 g of HOBt, dissolve them in 100 ml of DMF, cool the solution (≤10℃), add 11.36 g of pre-cooled DIC, stir and activate for 3-5 min. After activation, the amino acid solution temperature is 15±3℃. Add the activated solution to the swollen resin, then add 1.1 g of DMAP, and couple the reaction at 20-30℃ for 6.0 h. Stop the reaction, wash three times with DMF, the amino acid substitution degree is 0.72 mmol / g, and the reaction scale is 21.6 mmol.

[0114] Weigh 0.8g DMAP, 6.7g DIEA, and 4.5g Ac2O and dissolve them in 150ml DMF. Add the solution to a solid-phase column and perform a capping reaction for 2 hours. Wash with DMF 4 times.

[0115] Add 150 ml of DBLK solution (20% piperidine / DMF) for deprotection twice (5+15 min), wash 6 times with DMF, and ninhydrin test is positive. Weigh Fmoc-Pro-OH (14.57 g) and HOBt (7.00 g), add DMF (120 ml) to dissolve and cool (≤10℃), then add pre-cooled DIC (8.18 g), stir and activate for 3-5 min; after activation, the amino acid solution temperature is 15±3℃, add the activated solution to the swollen resin, and couple the reaction at 20-30℃ for 2.0 h. (Ninhydrin test is negative), stop the reaction, wash 4 times with DMF, 3 times with DCM, and 2 times with methanol, and vacuum dry. Cleavage by TFA:TIS:H2O = 92.5:2.5:2.5 (V / V / V), dry to obtain 13.47 g of product, yield 93%, purity 98.9%. The racemic impurity Fmoc-D-Pro-D-Ala-OH was 0.07%.

[0116] Example 22 Synthesis of Fmoc-Pro-D-Ala-OH

[0117] Weigh 100g of Fmoc-D-Ala-OH and 44.36g of HOSu, add them to DCM (800ml) and stir until dissolved and clear. Cool to 0℃. Weigh 79.53g of DCC and add it to DCM (500mL), stir to dissolve, cool to 0℃, and add it dropwise to the above solution. Stir the solution at 0℃ for 3 hours, and a white solid precipitates. Monitor the reaction progress by TLC.

[0118] After the reaction was complete, the reaction was stopped, filtered, and the solid was washed twice with DCM. The filtrates were combined. The filtrate was washed twice with water and once with saturated brine. It was then dried over anhydrous sodium sulfate. The organic layer was concentrated under vacuum until no liquid condensed. A mixed solvent (petroleum ether / isopropyl ether, V / V, 1 / 1) was added to the oily concentrate, and a white solid precipitated. The solid was filtered and dried under vacuum to obtain 124.58 g, with a yield of 95%.

[0119] Weigh 120g of Fmoc-D-Ala-OSu and dissolve it in THF (400mL), then cool to 0-5℃. Add 43.97g of H-Pro-OH and 40.48g of sodium carbonate to THF / water (V / V, 1 / 3, 800mL), stir to dissolve, and add dropwise to the above reaction solution. Stir and react at 0-5℃ for 1h, then heat to 20-25℃ and react for 5h. Monitor the reaction progress by TLC. After the reaction is complete, stop the reaction and extract twice with 1N hydrochloric acid at pH 5. Combine the organic layers, wash three times with 0.5N dilute hydrochloric acid, wash once with 300mL saturated brine, and dry with anhydrous sodium sulfate. Concentrate the organic layer until no condensate drips, recrystallize from isopropyl ether to obtain 187.42g, yield 87%, purity 99%, and racemic impurity Fmoc-D-Pro-D-Ala-OH 0.05%.

[0120] Comparative Example 1: Solid-phase synthesis of Degarelk

[0121] Following the above method for synthesizing degarelk, the starting resin was Rink Amide AM resin, and the deprotecting agent was replaced with a 20% piperidine / DMF solution. Amino acids were sequentially coupled according to the degarelk peptide sequence, with the 10th amino acid being Fmoc-D-Ala-OH and the 9th amino acid being Fmoc-Pro-OH. The synthesis scale was 30 mmol. The peptide resin was cleaved, purified, and lyophilized to obtain 18.48 g, with a yield of 40.9% and a purity of 99.1%. The impurity [4Aph(D / L-Hydantoinacetyl)] was excluded. 5 Degarelix was 0.12%, and impurities [D-Pro] were present. 9 Deagarelix was 0.16%.

[0122] Comparative Example 2: Solid-phase synthesis of Degarelk

[0123] Following the above-described synthetic method for degarelic peptides, the starting resin was Rink Amide AM resin, the deprotecting agent was replaced with a 20% morpholine / DMF solution, the 10th amino acid was Fmoc-D-Ala-OH, and the 9th amino acid was Fmoc-Pro-OH. Amino acids were sequentially coupled according to the degarelic peptide sequence. The synthetic scale was 30 mmol. The peptide resin was cleaved, purified, and lyophilized to obtain 18.87 g, with a yield of 41.3% and a purity of 99.3%. The impurity [4Aph(D / L-Hydantoinacetyl)] was excluded. 5 Degarelix was 0.09%, and impurities [D-Pro] were present. 9 Deagarelix was 0.14%.

[0124] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention, and should not be construed as a limitation of the present invention.

Claims

1. A method for preparing degarelk, characterized in that: Using Rink amino resin as the starting resin, starting from Fmoc-protected amino acids, a dipeptide fragment Fmoc-Pro-D-Ala-OH was introduced between the 9th and 10th positions. Solid-phase synthesis was adopted, and a peptide linking reaction was carried out with a coupling agent to sequentially link Fmoc-protected amino acids to obtain a protected decapeptide resin. The Fmoc groups of the amino acids were removed sequentially by the deprotecting reagent morpholine / DMF, and the crude peptide was obtained by cleavage with a lysis reagent. The crude peptide was dissolved, filtered, purified in one step, converted to salt in two steps, concentrated, and lyophilized to obtain the degarelc product. The main steps include: 1) Using Fmoc-protected Rink amino resin as the starting resin, the dipeptide fragment Fmoc-Pro-D-Ala-OH was introduced, and morpholine / DMF was used as the deprotection agent to couple it with Rink resin to obtain Fmoc-Pro-D-Ala-Rink resin. 2) Using the Fmoc solid-phase synthesis method, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph(Cbm)-OH, Fmoc-4Aph(Hor)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-4Cpa-OH and Fmoc-D-2Nal-OH were sequentially coupled with coupling agents according to the amino acid sequence of degarelcke, with morpholine / DMF as the de-Fmoc removal reagent during the process; 3) The N-terminal Fmoc of degarelk was removed using morpholine / DMF, followed by acetyl capping, washing, and drying to obtain peptide resin; 4) Cut the peptide resin with a cleavage reagent, precipitate, and centrifuge to obtain crude peptide; 5) The crude peptide was dissolved in water, acetonitrile, and acetic acid, and filtered through a filter membrane; it was purified in one step using the TEAP / acetonitrile system, and the fraction with a purity greater than 98% was collected; it was then converted to salt in two steps using ammonium acetate / acetonitrile and acetic acid / acetonitrile systems; after concentration and lyophilization, the Degarelk peptide was obtained.

2. The preparation method according to claim 1, characterized in that, The Rink amino resin is selected from Rink amide AM and Rink amide MBHA.

3. The preparation method according to claim 1, characterized in that, The degree of substitution of the amino resin is 0.40 to 0.90 mmol / g.

4. The preparation method according to claim 1, characterized in that, The Fmoc-Pro-D-Ala-OH can be synthesized by either solid-phase or liquid-phase methods.

5. The preparation method according to claim 1, characterized in that, The Fmoc-Pro-D-Ala-OH is synthesized by liquid phase.

6. The preparation method according to claim 1, characterized in that, The coupling agent is selected from carbodiimide type compounds and onium salt type compounds.

7. The preparation method according to claim 1, characterized in that, The coupling agent is selected from DIC / HOBt, EDCI / HOBt, HBTU / DIEA / HOBt, and TBTU / DIEA / HOBt.

8. The preparation method according to claim 1, characterized in that, The coupling agent is DIC / HOBt.

9. The preparation method according to claim 1, characterized in that, The deprotection reagent, morpholine / DMF, has a volume ratio concentration of 20%–50%.

10. The preparation method according to claim 1, characterized in that, The deprotecting agent, morpholine / DMF, has a volume ratio concentration of 20%.

11. The preparation method according to claim 1, characterized in that, The lysis reagent is selected from TFA / TIS / H2O, TFA / TIS, and TFA / H2O.

12. The preparation method according to claim 1, characterized in that, The lysis reagent is TFA / TIS / H2O.

13. The preparation method according to claim 4, characterized in that, The solid-phase synthesis of the dipeptide Fmoc-Pro-D-Ala-OH is characterized by using CTC resin or Wang resin as the starting resin, gradually coupling Fmoc-D-Ala-OH and Fmoc-Pro-OH, and obtaining Fmoc-Pro-D-Ala-OH after cleavage and recrystallization.

14. The preparation method according to claim 5, characterized in that, The liquid-phase synthesis of the dipeptide Fmoc-Pro-D-Ala-OH is characterized by the following steps: Fmoc-D-Ala-OH forms an active ester Fmoc-D-Ala-OSu under DCC / HOSu conditions, which then reacts with H-Pro-OH in a THF / H2O solvent under Na2CO3 conditions to generate the dipeptide Fmoc-Pro-D-Ala-OH. After recrystallization with isopropyl ether, the finished dipeptide is obtained. Using CTC resin or Wang resin as the starting resin, Fmoc-D-Ala-OH and Fmoc-Pro-OH are gradually coupled together, and after cleavage and recrystallization, Fmoc-Pro-D-Ala-OH is obtained.

15. The preparation method according to claim 1, characterized in that, In the acetyl-capping method described in step 3), the molar amounts of acetic anhydride and DIEA are 1.5 to 2.0 times that of the N-terminal free amino group.

16. The preparation method according to claim 1, characterized in that, The acetyl capping described in step 3) is carried out at 0–5°C.

17. The preparation method according to claim 1, characterized in that, The washing method described in step 3) is to wash with DMF and DCM in sequence.

Images