Solid-liquid synthesis process of high-purity leuprorelin

Abstract

The invention relates to the technical field of leuprorelin synthesis, in particular to a solid-liquid synthesis process of high-purity leuprorelin, Fmoc-Arg (Pbf)-OH, Fmoc-Tyr (tBu)-OH and 2-CTC resin are used as initial raw materials, and a leuprorelin crude product solid is obtained through a series of treatment such as deprotection, condensation, cutting, fragment docking, C-terminal dipeptide condensation and cracking. Compared with the prior art, the method has the remarkable advantages of simplicity and convenience in operation, low raw material investment, short production period, low cost and the like, meanwhile, the product synthesis yield is high (can reach 85%), the environmental pollution is small, industrial production is easy to realize, and the method has a wide market application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of leuprolide synthesis, in particular to a solid-liquid synthesis process of high-purity leuprolide. BACKGROUND

[0002] Leuprolide is also known as Leuprorelin, and its Chinese name is Yin Nantong or Leuprolide Acetate. Its chemical name is Pyr-His-Trp-Ser-Tyr-(D-Leu)-Leu-Arg-Pro-NHEt, its molecular formula is C 59 H 84 N 16 O 12 , and its molecular weight is 1209.45.

[0003] Leuprolide is a potent gonadotropin-releasing hormone (GnRH) agonist. It is a highly active analogue of luteinizing hormone-releasing hormone (LHRH) produced in the hypothalamus, which is a synthetic, water-soluble nonapeptide with both ends blocked. Leuprolide can stimulate the pituitary to secrete gonadotropin, which in turn stimulates the synthesis of sex steroids in the reproductive organs. However, long-term and large-dose use of leuprolide can desensitize the LHRH receptor, thereby inhibiting the secretion of gonadotropin and reducing the production of testicular or ovarian sex steroids. In clinical practice, leuprolide is widely used and is often used to treat endometriosis; it is suitable for uterine fibroids accompanied by menorrhagia, lower abdominal pain, back pain, and anemia; it is also effective for premenopausal breast cancer patients with positive estrogen receptors; it can also be used to treat prostate cancer and central precocious puberty.

[0004] The research and development process of leuprolide can be traced back to 1974, when the Japanese Takeda Pharmaceutical Fujino Masahiko research team first completed the discovery and synthesis process development of the compound. As a nonapeptide amide derivative of luteinizing hormone-releasing hormone, the compound exhibits significant biological activity, and the research team has applied for patent protection for the compound and its synthesis method in Japan (Patent No. JP19740027442), Germany (Patent No. DE2446005) and the United States (Patent No. US4008209). The synthesis process described in the patent uses tert-butyloxycarbonyl (Boc) protection strategy: first, Boc-Pro-OH is fixed on chloromethyl polystyrene resin (Merrifield resin) through triethylamine, and then each amino acid is connected step by step through solid-phase synthesis. After synthesis, amine hydrolysis is carried out using ethylamine at low temperature, the fully protected peptide is extracted with methanol, and after concentration and drying, the target product is finally obtained by removing the side chain protecting group with hydrogen fluoride (HF) gas. However, the Boc synthesis route has significant defects: high risk, strong acid and strong corrosion of trifluoroacetic acid (TFA) is required for each step of deprotection, and resin cleavage relies on highly toxic hydrogen fluoride (HF), which is extremely risky; environmental problems: a large amount of three wastes containing strong acid and fluoride are generated, which is difficult to handle; low process efficiency: low yield, high cost, and difficult to realize industrialized production. Domestic patent CN110386964A uses a step-by-step condensation method, which has a significant impact on yield. Each deprotection step uses piperidine, which is a precursor of a controlled substance, and the washing frequency and amount after each deprotection step are large, which is not suitable for large-scale production of short peptides. In addition, the patent does not solve the problem of dipeptide missing impurities well. Patent CN1865280A uses Wang resin, which not only produces a large amount of diketopiperazine (DKP) side reactions, but also produces impurities during cleavage, so it cannot be used for large-scale production. At the same time, this process also uses piperidine to remove Fmoc, involving precursor of a controlled substance and high cost.

[0005] The current market uses a chemical synthesis method to prepare leuprolide, which is mostly carried out by step-by-step condensation. This process has the following significant problems: first, the existing process can only achieve compound synthesis by solid-phase method, which not only requires a large amount of manpower, material resources and time cost, but also has a low reaction yield of about 40%, and produces a high content of related impurities; second, the reagent used in the deprotection step is piperidine, which is a precursor of a controlled substance, not only has high procurement cost, but also involves strict control requirements; third, the step-by-step condensation reaction process easily causes serious DKP side reactions, and the process is difficult to realize large-scale production; in addition, the reaction also produces C-terminal missing proline (Pro) and dipeptide impurities, etc. SUMMARY

[0006] The present application aims to provide a solid-liquid synthesis process of high-purity leuprolide to solve the problems in the above background.

[0007] To solve the above technical problems, the present application provides the following technical solutions: 1) adding a DMF solution of DIEA and Fmoc-Arg(Pbf)-OH to 2-CTC resin for reaction, then washing with DMF, adding a capping reagent to cap the resin, and obtaining Fmoc-Arg(Pbf)-2-CTC resin; 2) using a deprotection solution prepared by mixing dipropylamine and DMF at a volume ratio of 1:4 to deprotect Fmoc-Arg(Pbf)-2-CTC resin, washing with DMF, then gradually condensing Fmoc-Leu-OH and Fmoc-(D)Leu-OH, using a solution prepared by mixing TFE and DCM as a cleavage reagent to perform full-protection cleavage, adding n-heptane for dispersion, centrifuging to obtain full-protection peptide Fmoc-(D)Leu-Leu-Arg(Pbf)-OH powder, which is used as fragment 1; 3) adding a DMF solution of DIEA and Fmoc-Tyr(tBu)-OH to 2-CTC resin for reaction, then washing with DMF, adding a capping reagent to cap the resin, and obtaining Fmoc-Tyr(tBu)-2-CTC resin; 4) using a deprotection solution prepared by mixing dipropylamine and DMF at a volume ratio of 1:4 to deprotect Fmoc-Tyr(tBu)-2-CTC resin, washing with DMF, then gradually condensing Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-His(Trt)-OH, and Boc-Pyr-OH, using a solution prepared by mixing TFE and DCM as a cleavage reagent to perform full-protection cleavage, adding n-heptane for dispersion, centrifuging to obtain full-protection peptide Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH powder, which is used as fragment 2; 5) The fragment 1, fragment 2, Cl-HOBT and DIC are taken to carry out the ligation reaction, and the reaction solution is dispersed in pre-cooled MTBE, filtered, and then washed with MTBE beating; the ligation fragment is dissolved in DCM, HOBT and DIC are added, and then the dipeptide solution prepared by mixing dipeptide H-PRO-NHET.HCL, DCM and DIC is added after activation, and then the reaction is continued at elevated temperature; the reaction solution is added to pre-cooled MTBE to precipitate, MTBE is added for washing, and then filtered and dried under vacuum to obtain the fully protected leuprolide, i.e. Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-(D)leu-Leu-Arg(Pbf)-PRO-NHET; 6) A lysis solution is prepared by mixing TFA, Tis and distilled water, the lysis solution is pre-cooled, the fully protected leuprolide is weighed and added to the lysis solution to form a 1ml / g reaction solution, and then the reaction is continued at elevated temperature, and finally added to pre-cooled MTBE and stirred to precipitate, washed with MTBE beating, and dried to obtain the leuprolide crude solid.

[0008] Further, the substitution degree of the 2-CTC resin in steps 1) and 3) is 1.8-2.2mmol / g, the mass ratio of Fmoc-Arg(Pbf)-OH to DIEA is 1:2, the mass ratio of Fmoc-Tyr(tBu)-OH to DIEA is 1:2, and the amount of DMF is 10ml / g; the reaction conditions are that the reaction is carried out at a temperature of 18-22℃ for 2.5-3.5h; the capping reagent is prepared by mixing DMF, MEOH and DIEA in a volume ratio of 2:1:1; and the capping is carried out twice at 20-25℃, and each capping time is 1-1.5h.

[0009] Further, the deprotection times in steps 2) and 4) are 2 times, the first deprotection time is 5min, and the second deprotection time is 20min; the cleavage reagent is prepared by mixing TFE and DCM in a volume ratio of 1:4.

[0010] Further, the step-by-step condensation of Fmoc-Leu-OH and Fmoc-(D)Leu-OH in step 2) includes the following steps: Fmoc-Leu-OH and Cl-HOBT were added into DMF, and then DIEA was added after the dissolution was completed. After activation, they were added into the resin for reaction. Ninhydrin detection was performed after the reaction. After the colorless reaction, DMF was used for washing to obtain Fmoc-Leu-Arg(Pbf)-2-CTC resin; the obtained Fmoc-Leu-Arg(Pbf)-2-CTC resin was deprotected by using a deprotection solution prepared by mixing dipropylamine and DMF at a volume ratio of 1:4, and then washed with DMF; Fmoc-(D)Leu-OH and Cl-HOBT were taken, and then DIEA was added after the dissolution was completed. After activation, they were added into the resin for reaction. Ninhydrin detection was performed after the reaction. After the colorless reaction, DMF was used for washing. Dipropylamine and DMF prepared at a volume ratio of 1:4 were added for deprotection, and then DMF was used for washing. Methanol was used for shrinkage. After drying, Fmoc-(D)Leu-Leu-Arg(Pbf)-2-CTC resin was obtained.

[0011] Further, the mass ratio of Fmoc-Leu-OH, Cl-HOBT and DIEA was 1:1:1.4, and the mass ratio of Fmoc-(D)Leu-OH, Cl-HOBT and DIEA was 1:1:1.4. The amount of DMF was 10 ml / g. The activation condition was 8-12 min at 0-10 ℃, and the reaction condition was 85-95 min at 20-25 ℃. The deprotection times were 2 times, the first time was 5 min, and the second time was 20 min. The methanol shrinkage times were 2 times.

[0012] Further, the full protection cutting time in step 2) was 1-2 h, and the full protection cutting time in step 4) was 1.5-2.5 h.

[0013] Further, the step-by-step condensation of Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-His(Trt)-OH and Boc-Pyr-OH in step 4) included the following steps: S1: Fmoc-Ser(tBu)-OH and Cl-HOBT were taken, and then DIC was added after the dissolution was completed. After activation, they were added into the resin for reaction. Ninhydrin detection was performed after the reaction. After the colorless reaction, DMF was used for washing to obtain Fmoc-Ser(tBu)-Tyr(tBu)-2-CTC resin; S2: the obtained Fmoc-Ser(tBu)-Tyr(tBu)-2-CTC resin is subjected to deprotection by using a deprotection solution prepared by mixing dipropylamine and DMF at a volume ratio of 1:4, and is washed by using DMF; Fmoc-Trp(Boc)-OH and Cl-HOBT are taken, dissolved in DMF, and then DIC is added after activation, and then the mixture is added to the resin for reaction, and after the ninhydrin test shows no color, the resin is washed by using DMF, and is shrunk by using methanol, and is dried to obtain Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-2-CTC resin; The steps of S1 and S2 are repeated to obtain Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-2-CTC resin.

[0014] Further, the mass ratio of Fmoc-Ser(tBu)-OH, Cl-HOBT and DIC is 1:1:1.4, the mass ratio of Fmoc-Trp(Boc)-OH, Cl-HOBT and DIC is 1:1:1.4, the amount of DMF is 10 ml / g, the activation condition is 8-12 min at 0-10 ℃, the reaction condition is 85-95 min at 20-25 ℃, the deprotection times is 2 times, the first time is 5 min, the second time is 20 min, and the methanol shrinkage times is 2 times.

[0015] Further, the mass ratio of the fragment 1, the fragment 2, Cl-HOBT and DIC in the step 5) is 1.1:1:2:2.2, the reaction condition is 2.5-3.5 h at a temperature of 30-34 ℃, the mass ratio of the coupled fragment, HOBT and DIC is 1:1.5:1.6, the activation condition is 15-25 min at a temperature of 18-22 ℃, the mass ratio of the dipeptide and DIC added in the dipeptide solution is 1:1, the standing time is 8-12 min, the amount of the dipeptide solution added is 10 ml / g, the reaction condition after the dipeptide solution is added is 25-35 min at a temperature of 13-17 ℃, the temperature is 21-25 ℃ after being heated, and the continuous reaction time is 4.5-5.5 h, and the washing times of MTBE is 2-3 times.

[0016] Further, the volume ratio of TFA, Tis and distilled water in the step 6) is 90:5:5, the precooling condition is -20 ℃, the time is 0.5-1.5 h, the reaction condition is 25-35 min at a temperature of 13-17 ℃, the temperature is 23-27 ℃ after being heated, and the continuous reaction time is 1.5-2 h.

[0017] Compared with the prior art, the application has the following beneficial effects: 1. The solid-liquid synthesis process of high-purity leuprolide described in the application provides a high-efficiency, high-purity solid-liquid combined synthesis method of leuprolide, which is characterized by solid-phase synthesis fragments and liquid-phase interfacing fragments, completely avoiding the by-product diketopiperazine (DKP) and the incomplete impurities of dipeptide condensation; and the purity of the leuprolide obtained by the application is increased to 85%, which reduces the purification cost investment, improves the purification yield, and the synthesis yield can reach 80-90%.

[0018] 2. The solid-liquid synthesis process of high-purity leuprolide described in the application, the deprotection reagent is dipropylamine in the synthesis process, which does not involve easy-to-poison and has a lower price of 1 / 5 of the deprotection agent hexahydropyridine in other patents, and the washing times after deprotection are also reduced to half, greatly reducing the cost; the feeding amount in the production process can also greatly reduce the cost investment, and can meet the demand of high-purity crude product.

[0019] 3. The solid-liquid synthesis process of high-purity leuprolide described in the application, when scaled up, the fragments can be used as intermediate products, prepared in advance, and the synthesis and cleavage production cycle can be shortened to 2-3 days, so that the synthesis process can be industrialized. BRIEF DESCRIPTION OF DRAWINGS

[0020] Fig. 1 The process flow chart of the solid-liquid synthesis process of high-purity leuprolide of the application.

[0021] Fig. 2 The crude product chromatogram of the solid-liquid synthesis process of high-purity leuprolide of the application.

[0022] Fig. 3 The crude product peak value graph of the solid-liquid synthesis process of high-purity leuprolide of the application. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the application will be described in detail below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, not all. Based on the embodiments in the application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the application.

[0024] The abbreviations involved in the specification and claims are explained in detail in Table 1.

[0025] Table 1

[0026] In the following specific embodiments, 2-CTC resin: item number S20367, from Shanghai Yuanye Biotechnology Co., Ltd.; Fmoc-Arg(Pbf)-OH: Catalog No. S20423, from Shanghai Yuan Ye Biotechnology Co., Ltd.; DMF: Catalog No. CBEQ-4-109001-1000, from Shanghai Anpu Experimental Technology Co., Ltd.; DIEA: Catalog No. 7087-68-5, from Shanghai Kaisai Chemical Co., Ltd.; MEOH: Catalog No. M116115, from Shanghai Aladdin Biochem Technology Co., Ltd.; Fmoc-Leu-OH: Catalog No. S20343, from Shanghai Yuan Ye Biotechnology Co., Ltd.; Cl-HOBT: Catalog No. 26198-19-6, from Zhengzhou Huiju Chemical Co., Ltd.; Fmoc-(D)Leu-OH: Catalog No. S20344, from Shanghai Yuan Ye Biotechnology Co., Ltd.; TFE: Catalog No. WHXY-114, from Jinan Guochentai Fu Chemical Co., Ltd.; DCM: Catalog No. C0761510124, from Nanjing Chemical Reagent Co., Ltd.; n-Heptane: Catalog No. HZ1703, from Shanghai Yaoqian Chemical Co., Ltd.; Fmoc-Tyr(tBu)-OH: Catalog No. S20381, from Shanghai Yuan Ye Biotechnology Co., Ltd.; Fmoc-Ser(tBu)-OH: Catalog No. S20381, from Shanghai Yuan Ye Biotechnology Co., Ltd.; DIC: Catalog No. 40107, from Shanghai Covalent Chemical Technology Co., Ltd.; Fmoc-Trp(Boc)-OH: Catalog No. S42682, from Shanghai Yuan Ye Biotechnology Co., Ltd.; Fmoc-His(Trt)-OH: Catalog No. S20379, from Shanghai Yuan Ye Biotechnology Co., Ltd.; Boc-Pyr-OH: Catalog No. K48-1, from Hubei Widel Chemical Technology Co., Ltd.; MTBE: Catalog No. 23101, from Tianjin Boshigui Technology Development Co., Ltd.; HOBT: Catalog No. S30886, from Shanghai Yuan Ye Biotechnology Co., Ltd.; Dipeptide H-PRO-NHET.HCL: Catalog No. S73917, from Shanghai Yuan Ye Biotechnology Co., Ltd.; TFA: Catalog No. W10707, from Shanghai Yuan Ye Biotechnology Co., Ltd.; Tis: Product number BD0954, sourced from Hubei Baidu Chemical Co., Ltd.

[0027] Example: Figs. 1-3 As shown, this invention provides a technical solution for the solid-liquid synthesis process of high-purity leuprolide, the specific steps of which are as follows: Step 1: Fragment 1: Fmoc-(D)Leu-Leu-Arg(Pbf)-OH Using 2-CTC resin (substitution value 1.8 mmol / g) as the solid support, the resin was modified with Fmoc-Arg(Pbf)-OH; 1) Weigh 1.5 eq of Fmoc-Arg(Pbf)-OH, add 10 ml / g of DMF to dissolve it completely, add 3 eq of DIEA, and react at 20℃ for 3 h; then wash twice with DMF; add a capping agent of DMF, MEOH and DIEA in a volume ratio of 2:1:1 to cap the resin, and cap the resin at 25℃ for 1 h each time, for a total of two capping processes to obtain Fmoc-Arg(Pbf)-2-CTC resin; 2) The obtained Fmoc-Arg(Pbf)-2-CTC resin was deprotected twice at 25°C using a deprotection solution prepared by dipropylamine and DMF at a volume ratio of 1:4. The first deprotection was performed for 5 min and the second for 20 min. The resin was washed four times with DMF. 1.5 eq of Fmoc-Leu-OH and 1.5 eq of Cl-HOBT were weighed and dissolved in 10 ml / g of DMF. Then 2.1 eq of DIEA was added and activated at 5°C for 10 min. The solution was then added to the resin and reacted at 25°C for 90 min. After the ninhydrin test showed no color, the resin was washed twice with DMF to obtain the Fmoc-Leu-Arg(Pbf)-2-CTC resin. 3) The obtained Fmoc-Leu-Arg(Pbf)-2-CTC resin was deprotected twice at 25℃ using a deprotection solution prepared by dipropylamine and DMF at a volume ratio of 1:4. The first time was 5 min and the second time was 20 min. The resin was washed four times with DMF. 1.5 eq of Fmoc-(D)Leu-OH and 1.5 eq of Cl-HOBT were weighed and dissolved in 10 ml / g of DMF. Then 2.1 eq of DIEA was added and activated at 5℃ for 10 min. The solution was then added to the resin and reacted at 25℃ for 90 min. After the ninhydrin test showed no color, the resin was washed three times with DMF. The deprotection solution prepared by dipropylamine and DMF at a volume ratio of 1:4 was added and the resin was deprotected twice at 25℃. The first time was 5 min and the second time was 20 min. The resin was then washed four times with DMF, condensed twice with methanol, and dried. 4) cleavage of full protection at 25°C with a solution of TFE and DCM in a volume ratio of 1:4 for 1.5 h, dispersion with n-heptane, followed by centrifugation to obtain the full protection peptide Fmoc-(D)Leu-Leu-Arg(Pbf)-OH powder ready for use; Step 2: Fragment 2: Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH The 2-CTC resin (substitution value 1.8 mmol / g) was used as the solid support and the resin was modified with Fmoc-Tyr(tBu)-OH; 1) 1.5 eq of Fmoc-Tyr(tBu)-OH was weighed out, dissolved in 10 ml / g of DMF, 3 eq of DIEA was added, and the reaction was carried out at 20°C for 3 h; then the resin was washed twice with DMF; the resin was capped with a capping reagent consisting of DMF, MEOH and DIEA in a volume ratio of 2:1:1, and capping was carried out at 25°C for 1 h each time, for a total of two capping times, to obtain Fmoc-Tyr(tBu)-2-CTC resin; 2) The Fmoc-Tyr(tBu)-2-CTC resin obtained was deprotected twice with a deprotection solution consisting of dipropylamine and DMF in a volume ratio of 1:4 at 25°C, for 5 min and 20 min respectively, and washed four times with DMF; 1.5 eq of Fmoc-Ser(tBu)-OH and 1.5 eq of Cl-HOBT were weighed out, dissolved in 10 ml / g of DMF, and then 2.1 eq of DIC was added, and the mixture was added to the resin after activation at 5°C for 10 min, and the reaction was carried out at 25°C for 90 min; after the ninhydrin test showed no color, the resin was washed twice with DMF and twice with methanol, and then dried by suction to obtain Fmoc-Ser(tBu)-Tyr(tBu)-2-CTC resin; 3) The Fmoc-Ser(tBu)-Tyr(tBu)-2-CTC resin obtained was deprotected twice with a deprotection solution consisting of dipropylamine and DMF in a volume ratio of 1:4 at 25°C, for 5 min and 20 min respectively, and washed four times with DMF; 1.5 eq of Fmoc-Trp(Boc)-OH and 1.5 eq of Cl-HOBT were weighed out, dissolved in 10 ml / g of DMF, and then 2.1 eq of DIC was added, and the mixture was added to the resin after activation at 5°C for 10 min, and the reaction was carried out at 25°C for 90 min; after the ninhydrin test showed no color, the resin was washed twice with DMF and twice with methanol, and then dried by suction to obtain Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-2-CTC resin; 4) The obtained Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-2-CTC resin is deprotected twice at 25°C with a deprotection solution of dipropylamine and DMF prepared at a volume ratio of 1:4, the first time for 5 min and the second time for 20 min, washed with DMF for 4 times; 1.5 eq of Fmoc-His(Trt)-OH and 1.5 eq of Cl-HOBT are weighed, dissolved in 10 ml / g of DMF, and then 2.1 eq of DIC is added, added to the resin after activation at 5°C for 10 min, reacted at 25°C for 90 min, washed with DMF for 2 times after ninhydrin detection is colorless, shrank with methanol for 2 times, and dried to obtain Fmoc-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-2-CTC resin; 5) The obtained Fmoc-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-2-CTC resin is deprotected twice at 25°C with a deprotection solution of dipropylamine and DMF prepared at a volume ratio of 1:4, the first time for 5 min and the second time for 20 min, washed with DMF for 4 times; 1.5 eq of Boc-Pyr-OH and 1.5 eq of Cl-HOBT are weighed, dissolved in 10 ml / g of DMF, and then 2.1 eq of DIC is added, added to the resin after activation at 5°C for 10 min, reacted at 25°C for 90 min, washed with DMF for 2 times after ninhydrin detection is colorless, shrank with methanol for 2 times, and dried; 6) Full protection cleavage is carried out at 25°C with a solution prepared from TFE and DCM at a volume ratio of 1:4 for 2 h, dispersed with n-heptane, and then centrifuged to obtain full protection peptide Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH powder for standby; Step 3: fragment docking; 1) Docking of fragments 1 and 2: 1.1 mmol of fragment 1, 1 mmol of fragment 2, 2 mmol of Cl-HOBT and 2.2 mmol of DIC are taken, reacted at 32°C for 3 h, slowly added to pre-cooled MTBE for dispersion, filtered, and then washed with MTBE for 2 times; 2) Condensation of C-terminal dipeptide: weigh the docked fragment 1 mmol, add DCM to dissolve, then add 1.5 mmol of HOBT and 1.6 mmol of DIC, activate at 20°C for 20 min; weigh 2 mmol of dipeptide H-PRO-NHET.HCL, dissolve in DCM, then add 2 mmol of DIC, mix and stand for 10 min; then add 10 ml / g of dipeptide solution to the fragment solution, react at 15°C for 30 min, then raise the temperature to 23°C and continue to react for 5 h; 3) After the reaction is completed, the reaction solution is slowly added to the pre-cooled MTBE to precipitate, the precipitate is relatively sticky, the supernatant is separated, MTBE is added for washing 2 times, the precipitate will be in a dispersed state and easy to filter, after filtration, vacuum drying is performed to obtain the fully protected leuprolide, i.e. Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-(D)leu-Leu-Arg(Pbf)-PRO-NHET; Step 4: Cleavage: A cleavage solution is prepared by mixing TFA, Tis and distilled water in a volume ratio of 90:5:5, the cleavage solution is pre-cooled at -20°C for 1 h, 1 mmol of the fully protected leuprolide is weighed, the cleavage solution is added to form a 1 ml / g reaction solution, the reaction is carried out at 15°C for 30 min, then the temperature is raised to 25°C and the reaction is continued for 1.5 h, finally it is slowly added to the pre-cooled MTBE and stirred to precipitate, dispersed, washed with MTBE 3 times, and dried to obtain the leuprolide crude solid.

[0028] According to Fig. 2 and 3 it can be known that the yield of the leuprolide crude product is 82.369%, the main peak has no large impurities before it, the missing 2-peptide impurity before the main peak is solved, the peak with a retention time of 54.189 after the main peak is the Pro missing peak generated by the raw material, which can be solved by replacing the raw material, and the purity of the main peak can reach 85-90%.

[0029] It should be noted that, in this text, relational terms such as first and second are used merely to distinguish one entity or action from another, and do not necessarily require or imply that there is any such actual relationship or order between these entities or actions. Moreover, the terms "include", "contain" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process method article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or includes elements inherent to such process method article or equipment.

[0030] It will be apparent to those skilled in the art that the application is not limited to the details of the above-exemplified embodiments and that the present application can be implemented in other particular forms without departing from the spirit or essential characteristics of the present application. The embodiments should therefore be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the above description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. No reference signs in the claims should be considered as limiting the scope of the claims with respect to the figures of the patent document.

Claims

1. A solid-liquid synthesis process for high-purity leuprolide, characterized in that: Includes the following steps: 1) Add DIEA and DMF solution of Fmoc-Arg(Pbf)-OH to 2-CTC resin for reaction, then wash with DMF, and then add end-capping reagent to end-cap the resin to obtain Fmoc-Arg(Pbf)-2-CTC resin. 2) The Fmoc-Arg(Pbf)-2-CTC resin was deprotected using a deprotection solution prepared by dipropylamine and DMF at a volume ratio of 1:

4. After washing with DMF, Fmoc-Leu-OH and Fmoc-(D)Leu-OH were gradually condensed. Then, a solution prepared by TFE and DCM was used as the cleavage reagent for fully protected cleavage. After dispersing with n-heptane and centrifugation, the fully protected peptide Fmoc-(D)Leu-Leu-Arg(Pbf)-OH powder was obtained and used as fragment 1. 3) Add DIEA and DMF solution of Fmoc-Tyr(tBu)-OH to 2-CTC resin for reaction, then wash with DMF, and then add end-capping reagent to end-cap the resin to obtain Fmoc-Tyr(tBu)-2-CTC resin. 4) The Fmoc-Tyr(tBu)-2-CTC resin was deprotected using a deprotection solution prepared with dipropylamine and DMF at a volume ratio of 1:

4. After washing with DMF, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-His(Trt)-OH, and Boc-Pyr-OH were gradually condensed. Then, a solution prepared with TFE and DCM was used as the cleavage reagent for fully protected cleavage. The peptide was dispersed in n-heptane and centrifuged to obtain the fully protected peptide Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH powder, which was used as fragment 2. 5) Take fragment 1, fragment 2, Cl-HOBT and DIC for docking reaction. Add the reaction solution to pre-cooled MTBE for dispersion, filter, and then add MTBE to slurry and wash. Take the docked fragment and dissolve it in DCM. Then add HOBT and DIC for activation. Then add the dipeptide solution made by mixing dipeptide H-PRO-NHET.HCl, DCM and DIC and letting it stand. Then raise the temperature to continue the reaction. The reaction solution was added to pre-cooled MTBE to precipitate the precipitate. After washing with MTBE, the solution was filtered and then dried under vacuum to obtain fully protected leuprolide, namely Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-(D)leu-Leu-Arg(Pbf)-PRO-NHET. 6) Prepare a lysis buffer by mixing TFA, Tis and distilled water. Pre-cool the lysis buffer, weigh out fully protected leuprolide, add it to the lysis buffer to form a 1 ml / g reaction solution, then heat up to continue the reaction. Finally, add it to pre-cooled MTBE and stir to precipitate. Wash with MTBE, dry under vacuum to obtain crude leuprolide solid.

2. The solid-liquid synthesis process for high-purity leuprolide according to claim 1, characterized in that: The degree of substitution of the 2-CTC resin mentioned in steps 1) and 3) is 1.8~2.2 mmol / g, the mass ratio of Fmoc-Arg(Pbf)-OH to DIEA is 1:2, the mass ratio of Fmoc-Tyr(tBu)-OH to DIEA is 1:2, and the amount of DMF used is 10 ml / g; the reaction conditions are at a temperature of 18~22℃ for 2.5~3.5 h; the end-capping reagent is prepared by DMF, MEOH and DIEA in a volume ratio of 2:1:1; the end-capping is carried out twice at 20~25℃, and the end-capping time is 1~1.5 h each time.

3. The solid-liquid synthesis process for high-purity leuprolide according to claim 1, characterized in that: The deprotection process described in steps 2) and 4) is repeated twice, with the first deprotection taking 5 minutes and the second deprotection taking 20 minutes; the cutting reagent is prepared by mixing TFE and DCM in a volume ratio of 1:

4.

4. The solid-liquid synthesis process for high-purity leuprolide according to claim 1, characterized in that: Step 2) describes the stepwise condensation of Fmoc-Leu-OH and Fmoc-(D)Leu-OH, which includes the following steps: Fmoc-Leu-OH and Cl-HOBT were dissolved in DMF, and then DIEA was added for activation. The mixture was then added to the resin for reaction. After ninhydrin detection showed no color, the resin was washed with DMF to obtain Fmoc-Leu-Arg(Pbf)-2-CTC resin. The obtained Fmoc-Leu-Arg(Pbf)-2-CTC resin was deprotected using a deprotection solution prepared by dipropylamine and DMF at a volume ratio of 1:4, followed by washing with DMF. Fmoc-(D)Leu-OH and Cl-HOBT were dissolved in DMF, and then DIEA was added for activation. The mixture was then added to the resin for reaction. After ninhydrin detection showed no color, the resin was washed with DMF, and then deprotected using a deprotection solution prepared by dipropylamine and DMF at a volume ratio of 1:4, followed by washing with DMF. The resin was then dried under vacuum after methanol shrinkage to obtain Fmoc-(D)Leu-Leu-Arg(Pbf)-2-CTC resin.

5. The solid-liquid synthesis process for high-purity leuprolide according to claim 4, characterized in that: The mass ratio of Fmoc-Leu-OH, Cl-HOBT, and DIEA is 1:1:1.4, the mass ratio of Fmoc-(D)Leu-OH, Cl-HOBT, and DIEA is 1:1:1.4, and the amount of DMF used is 10 ml / g; the activation conditions are 8-12 min at 0-10℃, and the reaction conditions are 85-95 min at 20-25℃; the deprotection is performed twice, with the first time for 5 min and the second time for 20 min; the methanol shrinkage is performed twice.

6. The solid-liquid synthesis process for high-purity leuprolide according to claim 1, characterized in that: The full protection cutting time mentioned in step 2) is 1~2 hours; the full protection cutting time mentioned in step 4) is 1.5~2.5 hours.

7. The solid-liquid synthesis process for high-purity leuprolide according to claim 1, characterized in that: Step 4) describes the stepwise condensation of Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-His(Trt)-OH, and Boc-Pyr-OH, which includes the following steps: S1: Take Fmoc-Ser(tBu)-OH and Cl-HOBT, add DMF to dissolve them, then add DIC to activate them, and then add them to the resin to react. After the ninhydrin test is colorless, wash with DMF to obtain Fmoc-Ser(tBu)-Tyr(tBu)-2-CTC resin. S2: The obtained Fmoc-Ser(tBu)-Tyr(tBu)-2-CTC resin was deprotected using a deprotection solution prepared by dipropylamine and DMF at a volume ratio of 1:4, and washed with DMF; Fmoc-Trp(Boc)-OH and Cl-HOBT were dissolved in DMF, and then DIC was added for activation before being added to the resin for reaction. After ninhydrin detection showed no color, the resin was washed with DMF, shrunk with methanol, and dried to obtain Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-2-CTC resin; Repeat steps S1 and S2 to obtain Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-2-CTC resin.

8. The solid-liquid synthesis process for high-purity leuprolide according to claim 7, characterized in that: The mass ratio of Fmoc-Ser(tBu)-OH, Cl-HOBT, and DIC is 1:1:1.4, the mass ratio of Fmoc-Trp(Boc)-OH, Cl-HOBT, and DIC is 1:1:1.4, the amount of DMF used is 10 ml / g, the activation conditions are 0~10℃ for 8~12 min, the reaction conditions are 20~25℃ for 85~95 min, the deprotection is performed twice, the first time for 5 min and the second time for 20 min, and the methanol shrinkage is performed twice.

9. The solid-liquid synthesis process for high-purity leuprolide according to claim 1, characterized in that: In step 5), the mass ratio of fragment 1, fragment 2, Cl-HOBT, and DIC is 1.1:1:2:2.2, and the reaction conditions are 30-34℃ for 2.5-3.5 h. After docking, the mass ratio of fragment, HOBT, and DIC is 1:1.5:1.6, and the activation conditions are 18-22℃ for 15-25 min. The mass ratio of dipeptide to DIC added to the dipeptide solution is 1:1, and the standing time is 8-12 min. The amount of dipeptide solution added is 10 ml / g, and the reaction conditions after adding the dipeptide solution are 13-17℃ for 25-35 min, followed by a temperature increase to 21-25℃, and a further reaction time of 4.5-5.5 h. The number of washes after adding MTBE is 2-3.

10. The solid-liquid synthesis process for high-purity leuprolide according to claim 1, characterized in that: In step 6), the volume ratio of TFA, Tis, and distilled water is 90:5:

5. The pre-cooling condition is -20℃, the time is 0.5~1.5h, the reaction conditions are 13~17℃ for 25~35min, the temperature after heating is 23~27℃, and the reaction time continues for 1.5~2h.

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