Liquid phase preparation method for semaglutide

Abstract

Provided is a liquid phase preparation method for semaglutide, which method mainly comprises: deprotecting compound C2 to obtain semaglutide C3, wherein R1 is H or Fmoc, and the remaining amino acids each carry or do not carry a protecting group; and a deprotection reagent is selected from one or more of piperazine and piperazine derivatives. The method for removing Fmoc by using the piperazine or piperazine derivatives results in few impurities. In particular, the Asp / Asn-related impurities, namely [iso-Asp9]-semaglutide and [D-Asp9]-semaglutide, are both present at low levels. Moreover, the method allows for direct purification without precipitation, such that the impurities can be substantially removed by means of simple purification, which is greatly conducive to industrial production.

Description

A liquid-phase preparation method for smegglutinin Technical Field

[0001] This invention relates to the field of drug preparation, and specifically to a method for preparing smegglutinin. Background Technology

[0002] Semaglutide, developed by Novo Nordisk of Denmark, is a long-acting glucagon-like peptide-1 (GLP-1) analogue with blood sugar-lowering, weight-loss, and cardiovascular protective effects. It received FDA approval in December 2017. In its structure, the Lys side chain at the N-terminus 20 position is modified with PEG, Glu, and 18C-dicarboxylic acid, significantly increasing its hydrophilicity and enhancing its binding affinity to albumin. Simultaneously, the Ala mutation at the N-terminus 2 position to Aib effectively prevents inactivation by DPP-IV enzymatic degradation. Patients only need weekly injections. Oral formulations of this drug are also currently available.

[0003] The CAS number for smegglutinin is 910463-68-2. The peptide sequence is as follows:

[0004] His 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Val 10 -Ser 11 -Ser 12 -Tyr 13 -Leu 14 -Glu 15 -Gly 16 -Gln 17 -Ala 18 -Ala 19 -Lys 20 (AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu 21 -Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Val 27 -Arg 28 -Gly 29 -Arg 30 -Gly 31 -OH

[0005] Among the existing semi-synthetic methods for preparing smegglutinin, patents CN115322250A and CN115197312B disclose methods similar to Arg... 34 The dipeptide Boc-His(PG)-Aib-OH conjugated with GLP-1(9-37) requires TFA or strong acid solvents for Boc protection removal, which is complex, highly toxic, consumes a large amount of solvent, and has a long precipitation cycle; in addition, it generates a large amount of waste liquid, resulting in poor environmental performance. Patents CN 110041399B and CN 113801233A disclose a 20% piperidine / DMF solution as the reagent for removing Fmoc protection from the main chain. However, during the piperidine deprotection process, the strong alkalinity easily causes the generation of peptide-related impurities, resulting in low molar yield, which is not conducive to large-scale industrial production.

[0006] The hazards of peptide-related impurities mainly include their impact on the stability and efficacy of drug formulations. For example, amide hydrolysis impurities can lead to peptide aggregation, a common problem in peptide API production and formulation. Aggregated peptides may form insoluble substances, reducing the stability of the drug formulation and potentially triggering allergic reactions or decreased efficacy. During synthesis and storage, peptides may form structural impurities due to acid loss, amino acid insertion, residual protecting groups, oxidation / reduction, etc. These impurities may cause peptide drug denaturation, thus failing to achieve therapeutic effects and instead causing toxic side effects. Some impurities, such as diastereopters, side chains, and chain-terminal impurities, may have immunogenicity, thereby affecting drug safety and efficacy. Racemization of chiral amino acid residues can have a devastating impact on the biological activity of peptides. For example, the formation of aspartimide may lead to the loss of stereochemical information in the peptide, thereby affecting the drug's biological activity. Therefore, strictly controlling the formation and levels of impurities is crucial in the research and development and production of peptide drugs. Summary of the Invention

[0007] In view of the shortcomings of the existing methods for preparing semaglutide, and in order to overcome the problems of using highly toxic solvents, large amounts of solvent, generating large amounts of waste liquid, producing peptide-related impurities, and low molar yield during the synthesis of semaglutide, this invention provides a technical solution for synthesizing semaglutide, which mainly includes the following steps:

[0008] A liquid-phase preparation method for smegglutinin mainly includes the following steps:

[0009] Deprotection of compound C2 yields smegglutinin C3:

[0010] R1 is H or Fmoc, and the remaining amino acids may or may not have protecting groups; the deprotecting agent is selected from one or more piperazines or piperazine derivatives.

[0011] Existing technologies all use piperidine for Fmoc deprotection. However, due to the strong basicity of piperidine, deprotection easily leads to Asp rearrangement to succinimide, followed by isomerization and ring opening, generating Asp / Asn-related impurities: [iso-Asp] 9 [Smegglutinin, [D-Asp] 9 The impurities semaglutide and semaglutide are similar in nature to the main component semaglutide and are difficult to remove during purification. The structures of these impurities are as follows:

[0012] The applicant unexpectedly discovered that the method of removing Fmoc using piperazine or piperazine derivatives produces fewer impurities, especially Asp / Asn-related impurities [iso-Asp]. 9 [Smegglutinin, [D-Asp] 9 Smeglucopyranosides are small in size and do not require precipitation, allowing for direct purification. Impurities can be largely removed through simple purification, which greatly benefits industrial production.

[0013] In some embodiments, the deprotecting agent is selected from one or more of piperazine, 1-methylpiperazine, 1-ethylpiperazine, 1-propylpiperazine, 1-(2-hydroxyethyl)piperazine, and piperazine-1-carboxylic acid tert-butyl ester.

[0014] In some embodiments, the deprotecting agent is 1-methylpiperazine.

[0015] In some embodiments, compound C2 is prepared by the following method: using Arg 34 Starting with compound C1 of GLP-1(9-37), the terminal amino group was modified with Fmoc-His(R1)-Aib-OH, and then the compound C2 was obtained by liquid-phase acylation coupling reaction with condensing agent and activator in alkaline solvent.

[0016] R1 is H or Fmoc, and the other amino acids may or may not have a protecting group.

[0017] In some embodiments, semaglutide C3 is prepared by the following method:

[0018] R1 is H or Fmoc, and the other amino acids may or may not have a protecting group.

[0019] In some embodiments, compound C2 is prepared by the following method: using compound Arg34 Starting with GLP-1(8-37), the terminal amino group was modified with Fmoc-His(R1)-OH, and then the compound C2 was obtained by liquid-phase acylation coupling reaction with a condensing agent and an activator in an alkaline solvent.

[0020] R1 is H or Fmoc, and the other amino acids may or may not have a protecting group.

[0021] In some embodiments, the reaction solvent in the preparation method of compound C2 is water or an organic solvent miscible with water.

[0022] In some embodiments, the organic solvent is selected from one or more of acetonitrile, DMF, NMP, and THF.

[0023] In some embodiments, the organic solvent accounts for 40% to 80%.

[0024] In some embodiments, in the preparation method of compound C2, the basic reagent is selected from one or more of DIEA, triethylamine, trimethylamine, carbonate, and phosphate.

[0025] In some embodiments, the amount of the deprotecting agent is 20 to 100 times that of Fmoc-His(R1)-Aib-OH and Fmoc-His(R1)-OH.

[0026] In some embodiments, the amount of the deprotecting agent piperazine is 40 times that of Fmoc-His(R1)-Aib-OH.

[0027] In some embodiments, the amount of the deprotecting agent 1-methylpiperazine is 40 times that of Fmoc-His(R1)-Aib-OH.

[0028] In some embodiments, the method for preparing smegglutinin can be a stepwise reaction or a one-pot reaction.

[0029] In some embodiments, the condensing agent is selected from one or more of DIC, DCC, and EDCI.

[0030] In some embodiments, the activator is selected from one or more of HOBt, HOPfp, and HOPO.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The method of this invention uses piperazine or piperazine derivatives to remove Fmoc, which greatly and simultaneously inhibits / reduces [iso-Asp]. 9 [Smegglutinin, [D-Asp] 9The formation of two related peptide impurities, [iso-Asp], in the crude peptide... 9 The content of [D-Asp]-semaglutide does not exceed 0.42%; 9 The content of smegglutinin does not exceed 0.43%.

[0033] On February 21, 2023, the Center for Drug Evaluation of the National Medical Products Administration issued Announcement No. 12 of 2023, entitled "Technical Guidelines for Pharmaceutical Research of Chemically Synthesized Peptide Drugs (Trial Implementation)," which sets the reporting limit, identification limit, and quality control limit for peptide-related impurities at 0.1%, 0.5%, and 1.0%, respectively. In the crude peptide of this invention, [iso-Asp... 9 [Smegglutinin, [D-Asp] 9 The content of smegglutinin has met the requirements of the identification limit and quality control limit.

[0034] The preparation method of this invention significantly reduces the difficulty of crude peptide purification and greatly improves the purity and yield of smegglutinin. After simple purification steps, these impurities in smegglutinin are basically removed, and the final purity of the refined peptide is above 99.3%; the maximum single impurity content does not exceed 0.16%, which is almost negligible; and the total molar yield is above 78.00%. Further purification further improves the quality of smegglutinin.

[0035] The method of this invention has the advantages of good synthesis effect, high purity, few impurities, high yield and low cost, which reduces the synthesis cost and is conducive to large-scale industrial production.

[0036] The method of this invention features mild reaction conditions, requires no sedimentation, has a simple process operation, a short production cycle, and consumes less solvent, which is environmentally friendly, reduces costs, and is conducive to industrial production. Attached Figure Description

[0037] Figure 1 is the HPLC chromatogram of crude smegglutinin peptide from Example 1 of the present invention.

[0038] Figure 2 is the HPLC chromatogram of the smegglutinin peptide from Example 1 of the present invention.

[0039] Figure 3 is the HPLC chromatogram of the crude smegglutinin peptide from Example 2 of the present invention.

[0040] Figure 4 is the HPLC chromatogram of the smegglutinin peptide from Example 2 of the present invention.

[0041] Figure 5 is the HPLC chromatogram of the crude smegglutinin peptide from Example 3 of the present invention.

[0042] Figure 6 is the HPLC chromatogram of the smegglutinin peptide from Example 3 of the present invention.

[0043] Figure 7 is the HPLC chromatogram of the crude smegglutinin peptide from Example 4 of the present invention.

[0044] Figure 8 is the HPLC chromatogram of the smegglutinin peptide from Example 4 of the present invention.

[0045] Figure 9 is the HPLC chromatogram of the crude smegglutinin peptide from Example 5 of the present invention.

[0046] Figure 10 is the HPLC chromatogram of the smegglutinin peptide from Example 5 of the present invention.

[0047] Figure 11 is the HPLC chromatogram of crude smegglutinin peptide from Example 6 of the present invention.

[0048] Figure 12 is the HPLC chromatogram of the smegglutinin peptide from Example 6 of the present invention.

[0049] Figure 13 shows the HPLC chromatogram of crude smegglutinin from Comparative Example 1.

[0050] Figure 14 shows the HPLC chromatogram of smegglutinin peptide from Comparative Example 1.

[0051] Figure 15 shows the HPLC chromatogram of crude smegglutinin from Comparative Example 2.

[0052] Figure 16 shows the HPLC chromatogram of smegglutinin peptide from Comparative Example 2. Detailed Implementation

[0053] The present invention will be further described in detail below through embodiments, which are intended to illustrate the invention and not limit it. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications also fall within the protection scope of the present invention.

[0054] The abbreviations used in this invention have the following meanings:

[0055] DCC: N,N'-Dicyclohexylcarbodiimide

[0056] DIC: N,N'-Diisopropylcarbodiimide

[0057] EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0058] HOSu: N-hydroxysuccinimide

[0059] HOPFP: Pentafluorophenol

[0060] HOBt: 1-Hydroxybenzotriazole

[0061] HOPO: 2-hydroxypyridine-N-oxide

[0062] DMF: N,N'-Dimethylformamide

[0063] THF: Tetrahydrofuran

[0064] ACN: Acetonitrile

[0065] NMP: N-methylpyrrolidone

[0066] Unless otherwise specified, the explanations of relevant terms used in this invention shall adopt the conventional interpretations in the prior art.

[0067] Example 1

[0068] Preparation of smegglutinin

[0069] Weigh out the dipeptide Fmoc-His-Aib-OH (2.38 g, 5.14 mol, 4.0 eq) and HOPFP (0.96 g, 5.14 mmol, 4.0 eq), add 15 mL of DMF, maintain the temperature in an ice-water bath at 0–10 °C, add EDCI (0.99 g, 5.14 mmol, 4.0 eq) while stirring, and continue the reaction for 1.0–3.0 h.

[0070] Weigh C1 (5.0g, 1.28mmol, 1.0q), add DMF (70ml) and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 with DIEA, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until C1 reaction is complete.

[0071] Piperazine (8.9 g, 103 mmol, 80 eq) was added to the reaction solution to remove the Fmoc protecting group. The deprotection time was 1.0–3.0 h. After deprotection, the pH was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude semaglutide solution with a molar yield of 90.56% and a purity of 87.29%. The crude product was purified to obtain refined semaglutide peptide with a molar yield of 78.00%, a purity of 99.74%, and a maximum single impurity of 0.10%. The HPLC chromatogram and data of the crude semaglutide peptide are shown in Figure 1 and Table 1, respectively. The refined semaglutide peptide was purified to obtain the crude product, and the HPLC chromatogram and data are shown in Figure 2 and Table 2, respectively.

[0072] Table 1: Statistics of impurities related to crude peptide Asp / Asn in Example 1

[0073] Table 2: Statistics of Impurities Related to Asp / Asn Peptide in Example 1

[0074] Example 2

[0075] Preparation of smegglutinin

[0076] Weigh out Fmoc-His-Aib-OH (2.38 g, 5.14 mol, 4.0 eq) and HOPFP (0.96 g, 5.14 mmol, 4.0 eq), add 15 mL of NMP, maintain the temperature in an ice-water bath at 0–10 °C, add EDCI (0.99 g, 5.14 mmol, 4.0 eq) while stirring, and continue the reaction for 1.0–3.0 h.

[0077] Weigh C1 (5.0 g, 1.28 mmol, 1.0 q), add NMP (70 ml) and water (30 ml) sequentially, stir until homogeneous, maintain temperature at 10–30 °C, adjust pH to 9–10 with triethylamine, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until C1 reaction is complete. Add 1-ethylpiperazine (11.7 g, 103 mmol, 80 eq) to the reaction solution to remove the Fmoc protecting group, deprotection time is 1.0–3.0 h. After deprotection is complete, adjust pH to 8.0 ± 0.5 with glacial acetic acid, dilute with water, filter, and obtain crude smegglutide solution with a molar yield of 91.68% and purity of 90.95%. After purification, obtain refined smegglutide peptide with a molar yield of 82%, purity of 99.32%, and maximum single impurity of 0.16%. The HPLC chromatogram and data of crude smegglutide peptide are shown in Figure 3 and Table 3, respectively. The crude product was purified to obtain smeglucopyranoside peptide. The HPLC chromatogram and data are shown in Figure 4 and Table 4, respectively.

[0078] Table 3: Statistics of impurities related to crude peptide Asp / Asn in Example 2

[0079] Table 4: Statistics of Impurities Related to Asp / Asn Peptide in Example 2

[0080] Example 3

[0081] Preparation of smegglutinin

[0082] Weigh out Fmoc-His-Aib-OH (2.38 g, 5.14 mol, 4.0 eq) and HOPO (0.57 g, 5.14 mmol, 4.0 eq), add 15 mL of THF, maintain the temperature in an ice-water bath at 0–10 °C, add DIC (0.65 g, 5.14 mmol, 4.0 eq) with stirring, and continue the reaction for 1.0–3.0 h.

[0083] Weigh C1 (5.0g, 1.28mmol, 1.0q), add THF (70ml), water (30ml), DMF (70ml), and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 with saturated sodium carbonate solution, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until C1 reaction is complete.

[0084] 1-Methylpiperazine (51.5 g, 514 mmol, 400 eq) was added to the reaction solution to remove the Fmoc protecting group. The deprotection time was 1.0–3.0 h. After deprotection, the pH was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude semaglutide solution with a molar yield of 92.72% and a purity of 90.71%. The crude product was purified to obtain refined semaglutide peptide with a molar yield of 80%, a purity of 99.64%, and a maximum single impurity of 0.07%. The HPLC chromatogram and data of the crude semaglutide peptide are shown in Figure 5 and Table 5, respectively. The refined semaglutide peptide was purified to obtain the crude product, and the HPLC chromatogram and data are shown in Figure 6 and Table 6, respectively.

[0085] Table 5: Statistics of impurities related to crude peptide Asp / Asn in Example 3

[0086] Table 6: Statistics of Impurities Related to Asp / Asn Peptide in Example 3

[0087] Example 4

[0088] Preparation of smegglutinin

[0089] Weigh out Fmoc-His-Aib-OH (2.38 g, 5.14 mol, 4.0 eq) and HOPFP (0.96 g, 5.14 mmol, 4.0 eq), add 15 mL of DMF, maintain the temperature in an ice-water bath at 0–10 °C, add DCC (1.06 g, 5.14 mmol, 4.0 eq) with stirring, and continue the reaction for 1.0–3.0 h.

[0090] Weigh C1 (5.0g, 1.28mmol, 1.0q), add acetonitrile (70ml) and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 with DIEA, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until C1 reaction is complete.

[0091] 1-Methylpiperazine (10.3 g, 103 mmol, 80 eq) was added to the reaction solution to remove the Fmoc protecting group. The deprotection time was 1.0–3.0 h. After deprotection, the pH was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude semaglutide solution with a molar yield of 89.63% and a purity of 90.84%. The crude product was purified to obtain refined semaglutide with a molar yield of 81.00%, a purity of 99.61%, and a maximum single impurity of 0.07%. The HPLC chromatogram and data of the crude semaglutide are shown in Figure 7 and Table 7, respectively. The refined semaglutide was purified to obtain refined semaglutide, and the HPLC chromatogram and data are shown in Figure 8 and Table 8, respectively.

[0092] Table 7: Statistics of impurities related to crude peptide Asp / Asn in Example 4

[0093] Table 8: Statistics of Impurities Related to Asp / Asn Peptide in Example 4

[0094] Example 5

[0095] Preparation of smegglutinin

[0096] Weigh out Fmoc-His(Fmoc)-Aib-OH (3.52 g, 5.14 mol, 4.0 eq) and HOPFP (0.96 g, 5.14 mmol, 4.0 eq), add 15 mL of DMF, maintain the temperature in an ice-water bath at 0–10 °C, add EDCI (0.99 g, 5.14 mmol, 4.0 eq) while stirring, and continue the reaction for 1.0–3.0 h.

[0097] Weigh C1 (5.0g, 1.28mmol, 1.0q), add acetonitrile (70ml) and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 with DIEA, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until C1 reaction is complete.

[0098] The reaction solution was treated with 1-methylpiperazine (20.6 g, 206 mmol, 160 eq) to remove the Fmoc protecting group. The deprotection time was 1.0–3.0 h. After deprotection, the pH was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude semaglutide solution with a molar yield of 89.56% and a purity of 90.89%. The crude product was purified to obtain refined semaglutide peptide with a molar yield of 78.00%, a purity of 99.41%, and a maximum single impurity of 0.10%. The HPLC chromatogram and data of the crude semaglutide peptide are shown in Figure 9 and Table 9, respectively. The refined semaglutide peptide was purified to obtain HPLC chromatogram and data, as shown in Figure 10 and Table 10, respectively.

[0099] Table 9: Statistics of Impurities Related to Crude Peptide Asp / Asn in Example 5

[0100] Table 10: Statistics of Impurities Related to Asp / Asn Peptide in Example 5

[0101] Example 6

[0102] Preparation of smegglutinin

[0103] Weigh out Fmoc-His-OH (1.90 g, 5.03 mol, 4.0 eq) and HOPO (0.56 g, 5.03 mmol, 4.0 eq), add 15 mL of DMF, maintain the temperature in an ice-water bath at 0–10 °C, add EDC·HCl (0.96 g, 5.03 mmol, 4.0 eq) while stirring, and continue the reaction for 1.0–3.0 h.

[0104] Weigh Arg 34 GLP-1 (8-37) (5.0 g, 1.26 mmol, 1.0 q) was added sequentially to DMF (70 ml) and water (30 ml), and stirred until homogeneous. The temperature was controlled at 10–30 °C, and the pH was adjusted to 9–10 using DIEA. The mixture was stirred until dissolved, and the activated dipeptide was slowly added to the solution, stirring until Arg was reached. 34 GLP-1 (8-37) reaction complete.

[0105] Piperazine (17.3 g, 201 mmol, 160 eq) was added to the reaction solution to remove the Fmoc protecting group. The deprotection time was 1.0–3.0 h. After deprotection, the pH was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude semaglutide solution with a molar yield of 88.26% and a purity of 89.93%. The crude product was purified to obtain refined semaglutide peptide with a molar yield of 81.00%, a purity of 99.68%, and a maximum single impurity of 0.08%. The HPLC chromatogram and data of the crude semaglutide peptide are shown in Figure 11 and Table 11, respectively. The refined semaglutide peptide was purified to obtain the crude product, and the HPLC chromatogram and data are shown in Figure 12 and Table 12, respectively.

[0106] Table 11: Statistics of impurities related to crude peptide Asp / Asn in Example 6

[0107] Table 12: Statistics of Impurities Related to Asp / Asn Peptide in Example 6

[0108] Comparative Example 1

[0109] Preparation of smegglutinin

[0110] Weigh out Fmoc-His-Aib-OH (2.38 g, 5.14 mol, 4.0 eq) and HOPFP (0.96 g, 5.14 mmol, 4.0 eq), add 15 mL of DMF, maintain the temperature in an ice-water bath at 0–10 °C, add EDCI (0.99 g, 5.14 mmol, 4.0 eq) while stirring, and continue the reaction for 1.0–3.0 h.

[0111] Weigh C1 (5.0g, 1.28mmol, 1.0q), add acetonitrile (70ml) and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 with DIEA, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until C1 reaction is complete.

[0112] Piperidine (8.76 g, 102 mmol, 80 eq) was added to the reaction solution to remove the Fmoc protecting group. The deprotection time was 1.0–3.0 h. After deprotection, the pH was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude semaglutide solution with a molar yield of 82.06% and a purity of 89.81%. The crude product was purified to obtain refined semaglutide peptide with a molar yield of 72.00%, a purity of 98.16%, and a maximum single impurity of 0.59%. The HPLC chromatogram and data of the crude semaglutide peptide are shown in Figure 13 and Table 13, respectively. The refined semaglutide peptide was purified to obtain the crude product, and the HPLC chromatogram and data are shown in Figure 14 and Table 14, respectively.

[0113] Table 13: Statistics of impurities related to crude peptide Asp / Asn in Comparative Example 1

[0114] Table 14: Statistics of Impurities Related to Asp / Asn in Comparative Example 1

[0115] Comparative Example 2

[0116] Preparation of smegglutinin

[0117] Weigh out Fmoc-His(Fmoc)-Aib-OH (3.52 g, 5.14 mol, 4.0 eq) and HOPFP (0.96 g, 5.14 mmol, 4.0 eq), add 15 mL of DMF, maintain the temperature in an ice-water bath at 0–10 °C, add EDCI (0.99 g, 5.14 mmol, 4.0 eq) while stirring, and continue the reaction for 1.0–3.0 h.

[0118] Weigh C1 (5.0g, 1.28mmol, 1.0q), add acetonitrile (70ml) and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 with DIEA, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until C1 reaction is complete.

[0119] Piperidine (8.76 g, 102 mmol, 80 eq) was added to the reaction solution to remove the Fmoc protecting group. The deprotection time was 1.0–3.0 h. After deprotection, the pH was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude semaglutide solution with a molar yield of 87.72% and a purity of 89.97%. The crude product was purified to obtain refined semaglutide peptide with a molar yield of 68.00%, a purity of 98.45%, and a maximum single impurity of 0.49%. The HPLC chromatogram and data of the crude semaglutide peptide are shown in Figure 15 and Table 15, respectively. The refined semaglutide peptide was purified to obtain the crude product, and the HPLC chromatogram and data are shown in Figure 16 and Table 16, respectively.

[0120] Table 15: Statistics of impurities related to crude peptide Asp / Asn in Comparative Example 2

[0121] Table 16: Statistics of Impurities Related to Asp / Asn in Comparative Example 2

[0122] As can be seen from the HPLC chromatograms and data in Examples 1-6 and the comparative examples, the preparation method of the present invention greatly and simultaneously inhibits / reduces [iso-Asp] 9 [Smegglutinin, [D-Asp] 9 The formation of two impurities, smegglutinin.

[0123] In the crude peptides of the comparative example, [iso-Asp] 9 [-Smegglutinin content exceeds 1.13%; [D-Asp] 9 The content of [iso-Aspeptide] exceeded 0.74%. However, surprisingly, in the crude peptides of Examples 1-6: 9 The content of [-smegglutinin] did not exceed 0.42%, a reduction of at least 62.83% compared to the control group; [D-Asp] 9 The content of smegglutinin does not exceed 0.43%, which is at least 41.89% lower than that of the control group.

[0124] In the comparative sample of sperm peptides, [iso-Asp] 9 [-Smegglutinin content exceeds 0.46%; [D-Asp] 9 The content of [iso-Aspeptide] exceeded 0.49%. However, surprisingly, in the crude peptides of Examples 1-6:9 The content of [-smegglutinin] does not exceed 0.05%, which is at least 89.13% lower than that of the control group; [D-Asp] 9 The content of smegraglutide in the controlled sample is no more than 0.08%, a reduction of at least 83.67% compared to the comparative example. The preparation method of this invention significantly reduces the difficulty of crude peptide purification, greatly improving the purity and yield of smegraglutide. After simple purification steps, these impurities in smegraglutide are essentially removed, resulting in a final peptide purity of over 99.3%; the maximum single impurity content is no more than 0.16%, which is almost negligible; and the total molar yield is over 78.00%. In contrast, the maximum single impurity content in the comparative example is no less than 0.49%.

[0125] In conclusion:

[0126] The method of this invention has the advantages of good synthesis effect, high purity, few impurities, high yield and low cost, which reduces the synthesis cost and is conducive to large-scale industrial production.

[0127] The method of this invention features mild reaction conditions, requires no sedimentation, has a simple process operation, a short production cycle, and consumes less solvent, which is environmentally friendly, reduces costs, and is conducive to industrial production.

Claims

1. A process for the liquid phase preparation of semaglutide, characterized in that, The main steps include: Compound C2 is deprotected to give semaglutide C3: Wherein, R1 is H or Fmoc, and the remaining amino acids may or may not have protecting groups; the deprotecting reagent is selected from one or more piperazines or piperazine derivatives.

2. The method of claim 1, wherein, The deprotecting agent is selected from one or more of piperazine, 1-methylpiperazine, 1-ethylpiperazine, 1-propylpiperazine, 1-(2-hydroxyethyl)piperazine, and piperazine-1-carboxylic acid tert-butyl ester.

3. The method of claim 1, wherein, The deprotecting agent is 1-methylpiperazine.

4. The method of claim 1, wherein, The compound C2 was prepared by the following method: using Arg 34 Starting with compound C1 of GLP-1(9-37), the terminal amino group was modified with Fmoc-His(R1)-Aib-OH, and then the compound C2 was obtained by liquid-phase acylation coupling reaction with condensing agent and activator in alkaline solvent. R1 is H or Fmoc, and the other amino acids may or may not have a protecting group.

5. The method of claim 1, wherein, Smegglutinin C3 is prepared by the following method: R1 is H or Fmoc, and the other amino acids may or may not have a protecting group.

6. The method of claim 1, wherein, The compound C2 was prepared by the following method: using compound Arg 34 Starting with GLP-1(8-37), the terminal amino group was modified with Fmoc-His(R1)-OH, and then the compound C2 was obtained by liquid-phase acylation coupling reaction with a condensing agent and an activator in an alkaline solvent. R1 is H or Fmoc, and the other amino acids may or may not have a protecting group.

7. The method according to any of claims 4-6, characterized by, In the preparation method of compound C2, the reaction solvent consists of water or an organic solvent miscible with water.

8. The method of claim 7, wherein, The organic solvent is selected from one or more of acetonitrile, DMF, NMP, and THF.

9. The method of claim 7, wherein, The organic solvent accounts for 40% to 80%.

10. The method of any one of claims 4-6, wherein, In the preparation method of compound C2, the basic reagent is selected from one or more of DIEA, triethylamine, trimethylamine, carbonate, and phosphate.

11. The method of claims 4-6, wherein, The amount of the deprotecting agent used is 20 to 100 times that of Fmoc-His(R1)-Aib-OH and Fmoc-His(R1)-OH.

12. The method of claim 11, wherein, The amount of the deprotecting agent piperazine used is 40 times that of Fmoc-His(R1)-Aib-OH.

13. The method of claim 11, wherein, The amount of the deprotecting agent 1-methylpiperazine used is 40 times that of Fmoc-His(R1)-Aib-OH.

14. The method of claims 4-6, wherein, The method for preparing smegglutinin can be a stepwise reaction or a one-pot reaction.

15. The method according to any one of claims 4-6, characterized in that, The condensation reagent is selected from one or more of DIC, DCC, and EDCI.

16. The method of any one of claims 4-6, wherein, The activator is selected from one or more of HOBt, HOPfp, and HOPO.

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