Synthesis process of semeglutide impurity

Abstract

The invention provides a process route of a semeglutide impurity 9Assub-semeglutide, which comprises the following steps: firstly, constructing a side chain protected semeglutide resin, then selectively removing a protecting group on a 9-site Asp side chain on the resin, then carrying out ring closing to specifically convert Asp into an Assu structure, and then splitting the whole polypeptide sequence from the resin by using strong acid. According to the process, carboxyl of an Asp side chain is directionally dissociated by utilizing a specific method, the carboxyl and ortho-amino are subjected to directional cyclization by utilizing the structural trend of the carboxyl to obtain a product, the process is high in conversion rate, meanwhile, due to the fact that cyclization occurs in the last step, the influence on an Assu ring in the peptide chain construction process can be effectively avoided, the experimental method is simple and easy to implement, and the method is suitable for industrial production. The yield and the purity of the obtained product are very high, and a guarantee is provided for impurity research of the semeglutide and stable production of the product.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide synthesis, and particularly relates to a synthesis process of semaglutide impurities. Background Art

[0002] Semaglutide is a new generation of GLP-1 analog developed by Novo Nordisk of Denmark. Its main chain consists of 31 amino acids, and the side chain of lysine at position 20 is a branched chain structure composed of AEEA, glutamic acid, and octadecanedioic acid. The presence of this branched chain structure extends the half-life of the semaglutide molecule in vivo. The structure of this compound is as follows: 1His-2Aib-3Glu-4Gly-5Thr-6Phe-7Thr-8Ser-9Asp-10Val-11Ser-12Ser-13Tyr-14Leu-15Glu-16Gly-17Gln-18Ala-19Ala-20Lys(Ste-γGlu-AEEA-AEEA)-21Glu-22Phe-23Ile-24Ala-25Trp-26Leu-27Val-28Arg-29Gly-30Arg-31Gly-OH, with the molecular formula C187H291N45O59 and a molecular weight of 4113.58.

[0003] Semaglutide replaces and modifies some amino acid sites in the natural structure of GLP-1, which can not only overcome the disadvantages of the natural structure of GLP-1 but also retain or even improve the activity of the natural structure, achieving the effect of stable blood glucose control in vivo. Through a large number of theoretical and experimental proofs, some amino acid modules in the natural GLP-1 structure are irreplaceable. For example, His at position 1, Gly at position 4, and Asp at position 9. It is the existence of these conservative structures that ensures the stable activity of GLP-1.

[0004] There are mainly two types of methods for the synthesis of semaglutide. One is the chemical synthesis method, and the other is the combination of the chemical synthesis method and the biological method. During the process of drug development, production, and preservation, a series of process and stability impurities will be generated, mainly including deletion peptides, insertion peptides, mislinked peptides, epimeric peptides, and structurally rearranged polypeptides, etc. In the commercialization process of drugs, the directed synthesis and research of impurities play an indispensable role in the safety evaluation of drugs. Some impurities still lack effective synthesis means due to the high synthesis difficulty, which has caused obstacles to the further research of polypeptide drugs.

[0005] Asp at position 9 in the semaglutide structure is a very conservative amino acid. At the same time, Asp is an important site for generating impurities. During the synthesis, purification, and storage of the product, an Asp rearrangement side reaction occurs, generating the impurity 9Assu-semaglutide. The reaction formula is as follows:

[0006] Precisely because the probability and harmfulness of this impurity are both very high, during the development of semaglutide, it is necessary to conduct sufficient research on this impurity. Due to the great difficulty in synthesizing this impurity, it has become very difficult to locate and further study this impurity in the product.

[0007] To achieve the quality research and stable production of semaglutide, it is very important to develop a process capable of synthesizing this impurity. Summary of the Invention

[0008] The problem solved by the present invention is to provide a process route for synthesizing the impurity of this active pharmaceutical ingredient in view of the lack of a synthesis process for the key impurity of semaglutide.

[0009] The production of the product is mainly achieved through the following technical solutions. The characteristics are as follows: Using the solid-phase method to synthesize the overall polypeptide sequence with special protection on the Asp side chain, specifically removing the protecting groups to obtain a polypeptide with an exposed side chain, using corresponding cyclization conditions to perform cyclization on the resin to obtain the Assu structure, and cleaving the polypeptide from the resin and removing all protecting groups to obtain 9Assu-semaglutide.

[0010] The process steps mainly include the following content: Step 1, synthesize semaglutide (P)-resin with fully protected side chains; Step 2, selectively remove the protecting group on the carboxyl group of the Asp side chain at position 9 in the structure obtained in Step 1; Step 3, cyclize the exposed carboxyl group of the Asp side chain with the amino group on the amino acid at position 10 on the main chain; Step 4, cleave the polypeptide resin and remove all protecting groups to obtain the corresponding semaglutide impurity.

[0011] In Step 1, P represents different protecting groups on the side chain. The semaglutide with fully protected side chain is synthesized by solid-phase method, and its structure is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(P1)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-resin.

[0012] In Step 1, P1 is the protecting group on the carboxyl group of the Asp side chain, and All or 2-PhiPr can be selected. When All is used, Wang or CTC resin is preferred; when 2-PhiPr is selected, Wang resin is preferred.

[0013] During the construction of the polypeptide structure, Fmoc-Lys(OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-OH is selected as the overall polypeptide fragment unit; for the 9th amino acid, Fmoc-Asp(OAll)-OH or Fmoc-Asp(O-2-PhiPr)-OH is selected; for other amino acids, one of tBu, OtBu, Pbf, Trt is selected as the side chain protecting group.

[0014] Solid-phase synthesis uses de-Fmoc protection and condensation coupling as the basic reactions for constructing the polypeptide chain. During the construction of the polypeptide chain, HATU, HBTU, HCTU, DIC, PyBOP, PyAOP are used as condensation reagents, DIPEA, NMM, TEA are used as bases, and HOBt, HOAt, Oxyma are used as activation reagents. Piperidine or a DMF solution of DBU is selected for deprotecting Fmoc.

[0015] After the polypeptide sequence is constructed, All is removed with Pd(PPh3)4 or PdCl2, and TFA is selected for removing 2-PhiPr.

[0016] T3P, T4P, PyAOP, PyBOP, HATU, HBTU are selected as the cyclization reagents in Step 3, and DIEA or TEA is used as the base.

[0017] The polypeptide is cleaved from the resin and all protecting groups are removed. The cleavage reagent is a mixed component of concentrated TFA and scavengers. The content of TFA is 85-95%, and the scavengers include one or several of TIS, water, EDT, phenol, TES, and DODT.

[0018] The abbreviated product structure obtained is His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Assu-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Ste-γGlu-AEEA-AEEA)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0019] Purification is carried out by high performance liquid chromatography. C8 or C18 is selected as the chromatographic column packing, and acetic acid is selected as the mobile phase medium.

[0020] The in-process control of the whole reaction can adopt the resin ninhydrin color reaction method or the HPLC method.

[0021] The present invention constructs protected semaglutide impurities on the resin by solid-phase synthesis method, and then cuts the product with TFA. The reaction conversion rate is high, the synthesis process is very specific, and it is very conducive to the research and production of impurities. Detailed implementation mode

[0022] In order to explain the process technical scheme and its advantages in the present invention more clearly, the following further elaborates on the present invention in combination with examples. The specific examples described here are only used to explain the present invention and are not used to limit the present invention.

[0023] The abbreviation of the product obtained in the present invention is His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Assu-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Ste-γGlu-AEEA-AEEA)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0024] Abbreviation Annotation Wang resin p - Benzyloxybenzyl alcohol resin CTC resin 2 - Chlorotrityl resin AEEA Aminoethoxyethoxyacetic acid Ste Octadecanedioic acid Trt Trityl DIEA N,N - Diisopropylethylamine tBu tert - Butyl Pbf 2,2,4,6,7 - Pentamethyldihydrobenzofuran - 5 - sulfonyl 2 - PhiPr 1 - Methyl - 1 - phenylethyl All Allyl HOAt 1 - Hydroxy - 7 - azabenzotriazole HBTU Benzotriazol - N,N,N',N' - tetramethyluronium hexafluorophosphate PyAOP Pyridinium - 1 - yloxy - tripyrrolidinophosphonium hexafluorophosphate PyBOP Benzotriazol - 1 - yloxy - tripyrrolidinophosphonium hexafluorophosphate DMF N,N - Dimethylformamide MTBE Methyl tert - butyl ether NMM N - Methylmorpholine HOBt 1 - Hydroxybenzotriazole T3P 1 - Propylphosphonic anhydride T4P 1 - Butylphosphonic anhydride TFA Trifluoroacetic acid TIS Triisopropylsilane DBU 1,8 - Diazabicyclo[5.4.0]undec - 7 - ene DMAP 4 - Dimethylaminopyridine

[0025] Example 1: Synthesis of Fmoc-Gly-CTC resin.

[0026] Fmoc-Gly-OH (1.78 g, 6 mmol) and CTC resin (0.78 mmol / g, 3.85 g, 3 mmol) were added to 40 mL of DMF. The mixture was stirred and DIPEA (2.6 mL, 15 mmol) was added dropwise to the reaction solution. After the reaction solution was stirred at room temperature for 3 hours, methanol (4 mL) was added and the reaction continued for 15 minutes. Then the reaction solution was dried by suction, washed with DMF (80 mL * 5), and dried by suction again to obtain 4.5 g of Fmoc-Gly-CTC resin. The degree of substitution was measured to be 0.63 mmol / g by the spectrophotometer method.

[0027] Example 2: Synthesis of Fmoc-Gly-Wang resin.

[0028] Fmoc-Gly-OH (2.97 g, 10 mmol), DIC (1.26 g, 10 mmol) and Oxyma (1.42 g, 10 mmol) were added to DMF (50 mL). After stirring and dissolving, Wang resin (1.07 mmol / g, 4.67 g, 5 mmol) was added to the above reaction solution. After the reaction solution was stirred at room temperature for 3 hours, the reaction solution was dried by suction, washed with DMF (150 mL * 5), then acetic anhydride (0.7 mL), pyridine (0.6 mL) and DMF (10 mL) were added and the reaction continued for 2 hours. Then the reaction solution was dried by suction, washed with dichloromethane (150 mL x 3), and then washed with MTBE (150 mL x 2), and dried by suction to obtain 6 g of Fmoc-Gly-Wang resin. The degree of substitution was measured to be 0.82 mmol / g by the spectrophotometer method.

[0029] Example 3: Synthesis of Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OAll)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-CTC resin.

[0030] Suspend Fmoc-Gly-CTC resin (0.64 g, 0.4 mmol, 0.63 mmol / g) in DMF (10 mL). After swelling for 1 hour, drain the DMF. Add a DMF solution of 20% piperidine (20 mL) to the resin and react with stirring for 30 minutes. Then drain the reaction solution and wash with DMF (100 mL x 6) to obtain H-Gly-CTC resin.

[0031] Coupling and deprotection of the 30th amino acid Fmoc-Arg(Pbf)-OH: Add Fmoc-Arg(Pbf)-OH (0.52 g, 0.8 mmol), HOBt (0.11 g, 0.8 mmol) and DIEA (0.1 g, 0.8 mmol) to the above resin and stir. After adding HBTU (0.3 g, 0.8 mmol), stir and react for 1 hour. Detect that the reaction has ended by the ninhydrin method, continue to react for 10 minutes, then drain the reaction solution and wash with DMF (20 mL x 4) to obtain Fmoc-Arg(Pbf)-Gly-CTC resin. Add a DMF solution of 20% piperidine (20 mL) to the resin after coupling Fmoc-Arg(Pbf)-OH, react with stirring for 30 minutes, drain the reaction solution, and wash with DMF (20 mL x 6) to proceed to the next amino acid coupling reaction.

[0032] Continue to couple Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH according to the coupling conditions and deprotection conditions in the above steps to obtain H-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-CTC resin.

[0033] Coupling and deprotection of the 20th amino acid Fmoc-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-OH: Fmoc-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-OH (0.72 g, 0.6 mmol), HOBt (0.11 g, 0.6 mmol) and DIC (76 mg, 0.6 mmol) were added to 10 mL of DMF and stirred for activation for 30 minutes. The entire activated solution was added to the resin and stirred for about 2 hours. The reaction was detected to be complete by the ninhydrin method. After continuing the reaction for 30 minutes, the reaction solution was drained and washed with DMF (20 mL x 6). A 20% piperidine DMF solution (20 mL) was added to the resin. After stirring for 30 minutes, the reaction solution was drained and washed with DMF (20 mL x 6) before the next amino acid coupling reaction.

[0034] Coupling and deprotection of the 21st amino acid Fmoc-Ala-OH: Add Fmoc-Ala-OH (0.4 g, 1.2 mmol), HOBt (0.162 g, 1.2 mmol) and DIEA (0.155 g, 1.2 mmol) to the above resin and stir. Add HBTU (0.44 g, 1.15 mmol) and stir for 1 hour. Use the ninhydrin method to detect that the reaction has ended. Continue to react for 10 minutes and then drain the reaction solution. Wash with DMF (20 mL x 4) and set aside. Add 20% piperidine DMF solution (20 mL) to the resin and react for 30 minutes under stirring. Drain the reaction solution, wash with DMF (20 mL x 6) and then proceed to the next amino acid coupling reaction.

[0035] Connect Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OAll)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Boc-His(Trt)-OH according to the above coupling conditions and deprotection conditions, and then obtain Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OAll)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-CTC resin.

[0036] Example 4: Selective deprotection of the Asp side chain protecting group of Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OAll)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-CTC resin.

[0037] The resin was washed three times with chloroform to remove residual DMF. The resin was transferred into a reaction flask, and chloroform in an amount twice the volume of the resin was added. Meanwhile, Pd(PPh3)4 (132 mg, 0.33 g / mmol), NMM (15 mL) were added. After replacing N2, the reaction mixture was stirred for 3 hours, and then washed multiple times with a DMF solution of sodium diethyldithiocarbamate until the washing solution was colorless, obtaining Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-CTC resin.

[0038] Example 5: Synthesis of Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-CTC resin.

[0039] DMF was added to the resin obtained in Example 4. While stirring, PyAOP (0.63 g, 1.2 mmol) and HOBt (162 mg, 1.2 mmol) were added, and DIPEA (0.2 mL, 1.2 mmol) was added dropwise. After the addition was completed, the reaction flask was placed on a shaker and reacted for 64 hours, then the reaction was stopped. The reaction solution was dried by suction, washed with DMF (20 mL * 5) and then with methanol, and the reaction resin was dried by suction to obtain Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Assu-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-CTC resin.

[0040] Example 6: Synthesis of Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(O-2-PhiPr)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin.

[0041] Fmoc-Gly-Wang resin (0.49 g, 0.4 mmol, 0.82 mmol / g) was suspended in DMF (6 mL) and swollen for 1 hour, then dried by suction. A 20% piperidine in DMF solution (80 mL) was added to the resin, and the reaction was carried out with stirring for 30 minutes. Then the reaction solution was dried by suction, and washed with DMF (80 mL x 6) to obtain H-Gly-Wang resin.

[0042] Coupling and deprotection of the 30th amino acid Fmoc-Arg(Pbf)-OH: Add Fmoc-Arg(Pbf)-OH (0.52 g, 0.8 mmol), HOBt (0.11 g, 0.8 mmol) and DIEA (0.1 g, 0.8 mmol) to the above resin and stir. After adding HBTU (0.3 g, 0.79 mmol), stir and react for 1 hour. Use the ninhydrin method to detect that the reaction has ended. After continuing the reaction for 10 minutes, drain the reaction solution and wash with DMF (20 mL x 4) to obtain Fmoc-Arg(Pbf)-Gly-Wang resin. Add a 20% piperidine DMF solution (20 mL) to the resin after coupling Fmoc-Arg(Pbf)-OH. After reacting with stirring for 30 minutes, drain the reaction solution and wash with DMF (20 mL x 6) to obtain H-Arg(Pbf)-Gly-Wang resin for the next amino acid coupling reaction.

[0043] Connect Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH according to the above deprotection conditions and coupling conditions to obtain H-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin.

[0044] Coupling and deprotection of the 20th amino acid Fmoc-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-OH: Add Fmoc-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-OH (0.72 g, 0.6 mmol), HOBt (0.11 g, 0.6 mmol) and DIC (76 mg, 0.6 mmol) to 10 mL of DMF and stir to activate for 30 minutes. Then transfer the activation solution to the resin and stir and react for about 2 hours. Use the ninhydrin method to detect that the reaction has ended. After continuing the reaction for 30 minutes, drain the reaction solution and wash with DMF (20 mL x 6). Add a 20% piperidine DMF solution (20 mL) to the resin. After reacting with stirring for 30 minutes, drain the reaction solution and wash with DMF (20 mL x 6) before proceeding to the next amino acid coupling reaction.

[0045] Coupling and deprotection of the 21st amino acid Fmoc-Ala-OH: Fmoc-Ala-OH (0.4 g, 1.2 mmol), HOBt (0.162 g, 1.2 mmol) and DIEA (0.155 g, 1.2 mmol) were added to the above resin and stirred. After adding HBTU (0.44 g, 1.15 mmol), the mixture was stirred and reacted for 1 hour. The reaction was detected to be completed by the ninhydrin method. After continuing the reaction for 10 minutes, the reaction solution was dried by suction, washed with DMF (20 mL x 4), and then used. 20% piperidine in DMF solution (20 mL) was added to the resin. After reacting for 30 minutes with stirring, the reaction solution was dried by suction, washed with DMF (20 mL x 6), and then the next amino acid coupling reaction was carried out.

[0046] According to the above deprotection conditions and coupling conditions, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(O-2-PhiPr)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Boc-His(Trt)-OH were connected respectively to obtain Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(O-2-PhiPr)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin.

[0047] Example 7: Selective deprotection of Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(O-2-PhiPr)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin.

[0048] To the resin obtained in Example 6 was added a dichloromethane solution of 1% TFA. The reaction was stopped after stirring at room temperature for 30 minutes. The reaction solution was drained to obtain Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin.

[0049] Example 8: Synthesis of Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Assu-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin.

[0050] DMF was added to the resin obtained in Example 7. While stirring, T4P (0.43 g, 1.2 mmol) and DIPEA (0.2 mL, 1.2 mmol) were added dropwise to the reaction solution. After the addition was completed, the reaction flask was placed on a shaker and reacted for 64 hours, then the reaction was stopped. The reaction solution was dried by suction, washed with DMF (20 mL * 5) and then with methanol, and the reaction resin was dried by suction to obtain Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Assu-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin.

[0051] Example 9: Synthesis of His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Assu-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Ste-γGlu-AEEA-AEEA)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (9Assu-semaglutide crude product).

[0052] The resin obtained in Example 5 or Example 8 was added to a cooled reaction solution of TFA:TIS:H2O:thioanisole = 92.5:2.5:2.5:2.5 (10 mL). The temperature was raised to room temperature, and the reaction was carried out on a shaker for 2.5 hours and then stopped. The reaction solution was filtered, the solid resin was washed with TFA, the collected mother liquor was poured into cooled methyl tert-butyl ether, and the precipitated solid was filtered, washed 3 times with MTBE and then dried by suction to obtain 9Assu-semaglutide crude product.

[0053] The obtained 9Assu-semaglutide crude product solid was purified by preparative chromatography. Using C18 as the column packing, an acetonitrile / water system was adopted. Phase A was an aqueous solution of 0.1% acetic acid, and phase B was acetonitrile. The purification gradient was 10 - 30% in 25 minutes. Those meeting the purity requirements were collected together and freeze-dried to obtain 9Assu-semaglutide pure product with a purity of 98%.

[0054] The reaction conditions of the present invention are mild, the total yield is high, the reagents used are all low-cost reagents, the purity of the impurities of semaglutide obtained is relatively high, which is convenient for further product research.

Claims

1. The present invention provides a synthetic process for the important impurity 9-Assu semaglutide of semaglutide, and the process steps include the following: Step 1, synthesize the side-chain fully protected semaglutide (P)-resin; Step 2, selectively remove the protecting group on the carboxyl group of the 9-position Asp side chain in the structure obtained in Step 1; Step 3, perform directed cyclization of the exposed carboxyl group of the Asp side chain with the amino group on the 10-position amino acid on the main chain; Step 4, cleave the polypeptide resin and remove all protecting groups to obtain the 9-Assu semaglutide impurity.

2. According to the synthesis method of step 1 in claim 1, it is characterized in that: The side-chain fully protected semaglutide structure is synthesized by a solid-phase method. P represents different protecting groups on the side chain, and its complete structure is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(P1)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-Glu(OtBu)-Phe-Ile-Ala-Trp(tBu)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-resin.

3. According to the methods of Claims 1 and 2, Step 1 is characterized in that: P1 is the protecting group on the carboxyl group of the Asp side chain, preferably All or 2-PhiPr. When P1 is All, Wang or CTC resin is used as the starting resin; when P1 is 2-PhiPr, Wang resin is selected.

4. The method according to claims 1-3, characterized in that: During the construction of the polypeptide, Fmoc-Lys((OtBu)Ste-γGlu(OtBu)-AEEA-AEEA)-OH is selected as the polypeptide fragment unit and incorporated into the peptide chain; for the 9-position amino acid, Fmoc-Asp(OAll)-OH or Fmoc-Asp(O-2-PhiPr)-OH is selected; for other amino acids, one of tBu, OtBu, Pbf, Trt is selected as the side-chain protecting group.

5. The method according to claims 1-4, characterized in that: Solid-phase synthesis uses de-Fmoc protection and condensation coupling as the basic reactions for constructing the polypeptide chain. During the construction of the polypeptide chain, HATU, HBTU, HCTU, DIC, PyBOP, PyAOP are used as condensation reagents, DIPEA, NMM, TEA are used as bases, and HOBt, HOAt, Oxyma are used as activation reagents. Piperidine or a DMF solution of DBU is selected for de-Fmoc removal.

6. The synthesis method according to step 2 of claim 1, characterized in that: Pd(PPh3)4 or PdCl2 is selected for All removal, and low-concentration TFA is selected for 2-PhiPr removal.

7. The synthesis method according to step 3 of claim 1, characterized in that: T3P, T4P, PyAOP, PyBOP, HATU, HBTU are selected as cyclization reagents, and DIEA or TEA is used as the base.

8. The synthesis method according to step 4 of claim 1, characterized in that: The cleavage reagent is a mixed component of concentrated TFA and scavengers. The content of TFA is 85-95%, and the scavengers include one or several of TIS, water, EDT, phenol, TES, and DODT.

9. According to the method of claims 1-8, the abbreviated structure of the obtained product is His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Assu-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Ste-γGlu-AEEA-AEEA)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

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