A process for the preparation of solimycin at specific sites with dipeptide feed

Abstract

The present application belongs to the field of polypeptide synthesis, and relates to a method for preparing somatostatin at specific sites by feeding dipeptides. In the present application, at least one of the 30th Ala, 24th Ala, 21st Glu, 15th Asp, 11th Thr and 9th Glu is fed in the form of a dipeptide, so that racemization of the above six sites during solid-phase synthesis is avoided from the reaction mechanism, thereby avoiding the generation of difficult-to-separate impurities near the main peak during chromatographic separation of somatostatin, making the obtained product easy to purify, ensuring product quality, and greatly improving the purification yield.

Description

Technical Field

[0001] This invention relates to the field of polypeptide synthesis, and in particular to a method for preparing high-purity semaglutide by solid-phase synthesis using six dipeptide fragments. Background Technology

[0002] Semaglutide is an endogenous GLP-1 analog with its Lys side chain modified by AEEA, γ-Glu, and octadecanoic acid. Its structure is as follows:

[0003]

[0004] This product was developed by Novo Nordisk and is primarily used to treat type 2 diabetes, with significant effects on weight control. The original manufacturer's production process involves main-chain fermentation combined with side-chain chemical modification.

[0005] Currently, solid-phase synthesis (SPS) can effectively prepare semaglutide active pharmaceutical ingredient (API). However, compared to the original manufacturer's bio-fermentation process, racemic impurities of amino acids are unique to SPS. Therefore, controlling these impurities during SPS is crucial to ensuring consistency with the original product's quality. Commonly used condensing agent systems in SPS include HATU / HOBt / DIEA, HBTU / HOBt / DIEA, PyBOP / HOBt / DIEA, DIC / HOBt, DIC / HOAt, and DIC / Cl-HOBt. These condensing agents can effectively control racemization during SPS, but they cannot completely eliminate it as in bio-fermentation processes.

[0006] Patent CN201910677458.3 divides the synthesis of semaglutide into three stages. Positions 37-17 are synthesized using a conventional solid-phase synthesis process with stepwise condensation. Positions 16-11 are prepared with CTC resin to form a fully protected peptide, which is then condensed with the 37-17 peptide resin. Finally, the four amino acids at positions 10-7 are gradually condensed. This process solves the technical challenge of condensing the amino acids at positions 16-11 during solid-phase synthesis. The impurities addressed are classified as the deletion of amino acids at positions 16-11, not racemization.

[0007] Patent CN201910105996.5 also divides the 31 amino acids of the semaglutide backbone into three segments: a first segment of 12 amino acids, a second segment of 12 amino acids, and a third segment of 7 amino acids. Each segment is prepared as a fully protected peptide on CTC resin and then synthesized in a liquid-phase system. This route is a classic fragmentation method for solid-liquid synthesis of peptides. The main technical challenge it addresses is the difficulty in the later condensation of long-chain peptides. However, it also fails to address racemic impurities with properties similar to semaglutide.

[0008] Patent CN201811466181.1 divides the 31 amino acids of the semaglutide backbone into six segments. The first peptide segment sequence is amino acids 7-10 of the semaglutide sequence; the second peptide segment sequence is amino acids 11-15; the third peptide segment sequence is amino acids 16-22; the fourth peptide segment sequence is amino acids 23-28; the fifth peptide segment sequence is amino acids 29-32; and the sixth peptide segment sequence is amino acids 33-37. These six segments are collectively referred to as a fully protected peptide on CTC resin. Unlike patent CN201910105996.5, this patent does not perform segment condensation in the liquid phase after obtaining the six peptide segments, but rather performs segment-by-segment condensation on the resin. Using this process to prepare semaglutide can significantly improve the purity of the crude product, but the cost is high and it is not suitable for large-scale industrial production. In addition, it does not focus on controlling racemic impurities.

[0009] Currently, the FDA's quality control requirements for impurities in generic peptide drugs are: impurities not present in the original drug must not exceed 0.1% in the generic drug. The best solution to meet these requirements is to avoid the formation of these impurities during the synthesis process. The original drug uses a fermentation process, theoretically eliminating racemic amino acid impurities. Therefore, controlling racemic impurities during solid-phase synthesis is particularly important. Summary of the Invention

[0010] The purpose of this invention is to provide a solid-phase synthesis method for semaglutide that can avoid the generation of difficult-to-separate racemic impurities.

[0011] In a first aspect, the present invention provides a method for solid-phase synthesis of semaglutide, comprising the steps of:

[0012] (a) Using Wang resin or CTC resin as a solid support, condense with Fmoc-Gly-OH in the presence of a coupling agent to obtain an amino acid resin; then couple it with the corresponding amino acid precursor according to the peptide sequence of semaglutide, and feed it in the form of a dipeptide at at least one of the following sites: 30 Ala, 24 Ala, 21 Glu, 15 Asp, 11 Thr, and 9 Glu to obtain a fully protected semaglutide peptide resin.

[0013] (b) The semaglutide resin was pyrolyzed to obtain crude semaglutide.

[0014] In another preferred embodiment, in step (a), the semaglutide is coupled sequentially according to the following peptide sequence: Fmoc-Arg(pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ala-Trp(Boc)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Ala-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu). -Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-T hr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Aib-OH, Boc-His(Trt)-OH, and Ste-γ-Glu(AEEA-AEEA-OH)-OtBu(side chain).

[0015] In another preferred embodiment, in step (a), the initial substitution degree of the king resin or CTC resin is independently 0.4 to 0.5 mmol / g, or the substitution degree of Fmoc-Gly-Resin is 0.35 to 0.40 mmol / g.

[0016] In another preferred embodiment, in step (a), the coupling agent for each coupling reaction is independently selected from the group consisting of: HOBt / DIC, HOAt / DIC, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, PyBOP / DIPEA, TBTU / DIPEA, or combinations thereof.

[0017] In another preferred embodiment, in step (a), each coupling reaction independently includes one or more of the following features:

[0018] (1) The amino acid precursor is first de-Fmoc group removed before the reaction. Preferably, the de-Fmoc reagent is 20% piperidine / DMF (volume ratio).

[0019] (2) The molar ratio of resin to amino acid precursor is 1:2.0-3.5, preferably 1:2.5-3.0, and the amount of resin used is calculated based on its total degree of substitution;

[0020] (3) The molar ratio of the amino acid precursor to the coupling agent is 1:1-1.5, preferably 1:1.1-1.2;

[0021] (4) The reaction solvent is a mixed solution of DMF / DCM, preferably a mixed solution of DMF / DCM in a volume ratio of 1:1; and / or

[0022] (5) DIC or DCC activating coupling agent is also added to the reaction solution. Preferably, the molar ratio of DIC to coupling agent is 1-1.2:1.

[0023] In another preferred embodiment, in step (a), each coupling reaction independently includes one or more of the following features:

[0024] (1) The reaction is carried out in an inert gas atmosphere, preferably N2;

[0025] (2) The reaction temperature is 25±10℃, preferably 25±5℃;

[0026] (3) The reaction time is 1-6 hours, preferably 2-3 hours (subject to Kaiser test results); and / or

[0027] (4) After a reaction is completed, the reaction solution is separated, acetylated (e.g., acetic anhydride) reagent is added for acetylation, and the resin is washed to proceed to the next reaction or to end the reaction.

[0028] In another preferred embodiment, in step (b), the cleavage reagent is a TFA solution containing 5 to 10% by volume a scavenger, wherein the scavenger is selected from the group consisting of dithiothreitol, anisole, phenol, mercaptoethanol, water, and triisopropylsilane, or combinations thereof.

[0029] In another preferred embodiment, the method further includes the steps of: reversed-phase chromatography purification, salt conversion, and lyophilization to obtain semaglutide peptide.

[0030] In another preferred embodiment, the reversed-phase chromatographic purification may include one or more features selected from the group consisting of:

[0031] (a) The chromatographic column is C. 18 Chromatographic column;

[0032] (b) Mobile phase: Phase A: 50 mM hydrogen phosphate (pH 3.6 ± 0.2): acetonitrile = 9:1; Phase B: acetonitrile;

[0033] (c) Column temperature: 42±2℃; and / or

[0034] (d) Gradient elution.

[0035] In another alternative example, the reversed-phase chromatography purification includes the steps of:

[0036] (a) Primary purification: A C8 column was used, with mobile phase A being 40 mmol / L diammonium hydrogen phosphate / water at pH 8.0 and mobile phase B being acetonitrile, using gradient elution; the semaglutide main peak fraction was collected, and the fraction with a purity >97.0% was considered a qualified primary purity fraction; and

[0037] (b) Secondary purification: The qualified fraction of the first purity was purified by gradient elution using a C8 column with 0.1% acetic acid / water as phase A and pure acetonitrile as phase B. The main peak fraction of semaglutide was collected. The fraction with a purity >99.0% and a single impurity <0.1% was considered a qualified fraction of the second purity.

[0038] In a second aspect, the present invention also provides a raw material composition for solid-phase synthesis of semaglutide, the composition comprising:

[0039] Fmoc-Ala-Trp(Boc)-OH, Fmoc-Ala-Ala-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Asp(OtBu)-Val-OH, Fmoc-Thr(tBu)-Phe-OH, and Fmoc-Glu(OtBu)-Gly-OH.

[0040] In another preferred embodiment, the composition further comprises one or more selected from the group consisting of: Fmoc-Gly-OH, Fmoc-Arg-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Aib-OH, Boc-His(Trt)-OH, Ste-γ-Glu(AEEA-AEEA-OH)-OtBu(side chain), or combinations thereof.

[0041] In another preferred embodiment, the composition further includes: royal resin or CTC resin.

[0042] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0043] Through extensive and in-depth research, and through numerous screenings and tests, the inventors have developed a solid-phase synthesis method for semaglutide. To control the formation of six racemic impurities similar in properties to semaglutide during synthesis, this invention provides a solid-phase synthesis process that involves feeding at specific sites in the form of dipeptides. At least one of the following sites—30-Ala, 24-Ala, 21-Glu, 15-Asp, 11-Thr, and 9-Glu—is fed as a dipeptide, thus preventing the chiral carbons of these six amino acids from participating in the condensation reaction. This mechanistically avoids the formation of racemic impurities at these sites, significantly reducing purification difficulty while maximizing the control of raw material costs. This invention is based on this principle.

[0044] the term

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0046] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0047] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0048] As used herein, the term “room temperature” or “normal temperature” refers to a temperature of 4–40°C, preferably 25 ± 5°C.

[0049] Semaglutide

[0050] Semaglutide is the first and currently the only oral glucagon-like peptide-1 (GLP-1) receptor agonist that can be used to improve blood glucose levels in patients with type 2 diabetes.

[0051] The structure is as follows:

[0052] H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AE EA-AEEA-γGlu-17-carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0053] Difficult-to-separate racemic impurities

[0054] After extensive screening, the inventors discovered that the racemic impurities at the following sites are very similar to the peak values ​​of semaglutide in solid-phase synthesis: Ala at position 30, Ala at position 24, Glu at position 21, Asp at position 15, Thr at position 11, and Glu at position 9.

[0055] Racemic impurities at these sites are difficult to separate by methods such as preparative liquid chromatography, which puts great pressure on the purification of crude semaglutide prepared by solid-phase synthesis.

[0056] Raw material composition

[0057] The present invention also provides a raw material composition for solid-phase synthesis of semaglutide, comprising:

[0058] Fmoc-Ala-Trp(Boc)-OH, Fmoc-Ala-Ala-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Asp(OtBu)-Val-OH, Fmoc-Thr(tBu)-Phe-OH, and Fmoc-Glu(OtBu)-Gly-OH.

[0059] Preparation method

[0060] This invention provides a solid-phase synthesis method for semaglutide. By feeding the 30th Ala, 24th Ala, 21st Glu, 15th Asp, 11th Thr, and 9th Glu in the semaglutide peptide sequence as dipeptides, these sites do not directly participate in the reaction, thereby avoiding the generation of racemic impurities at these sites. This greatly reduces the purification difficulty while minimizing the types of dipeptide raw materials.

[0061] Specifically, the method includes the following steps:

[0062] (a) Using Wang resin or CTC resin as a solid-phase support, condensation is performed with Fmoc-Gly-OH in the presence of a coupling agent to obtain an amino acid resin; then, the corresponding amino acid precursors are sequentially coupled according to the peptide sequence of semaglutide. Preferably, the precursors can be Fmoc-Arg(pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ala-Trp(Boc)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Ala-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fm (a) Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Aib-OH, Boc-His(Trt)-OH, and Ste-γ-Glu(AEEA-AEEA-OH)-OtBu (side chain) to obtain a fully protected semaglutide peptide resin; (b) Cleavage the semaglutide peptide resin to obtain crude semaglutide. The structure of the fully protected semaglutide peptide resin is as follows: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(sidechain)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-Resin.

[0063] In another preferred embodiment, the initial substitution degree of the king resin or CTC resin described in step (a) is independently 0.4 to 0.5 mmol / g, or the substitution degree of Fmoc-Gly-Resin is 0.35 to 0.40 mmol / g.

[0064] In another preferred embodiment, in step (a), the coupling agent for each coupling reaction is independently selected from the group consisting of: HOBt / DIC, HOAt / DIC, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, PyBOP / DIPEA, TBTU / DIPEA, or combinations thereof.

[0065] In another preferred embodiment, in step (b), the cleavage reagent is a TFA solution containing 5 to 10% by volume a scavenger, wherein the scavenger is selected from the group consisting of dithiothreitol, anisole, phenol, mercaptoethanol, water, and triisopropylsilane, or combinations thereof.

[0066] In another preferred embodiment, the method further includes the steps of: reversed-phase chromatography purification, salt conversion, and lyophilization to obtain semaglutide peptide.

[0067] Preferably, the product prepared by the preparation method of the present invention can be purified using a specific chromatographic purification method.

[0068] For example, the reversed-phase chromatography purification may include one or more features selected from the group consisting of:

[0069] (a) The chromatographic column is a C18 column;

[0070] (b) Mobile phase: Phase A: 50 mM hydrogen phosphate (pH 3.6 ± 0.2): acetonitrile = 9:1; Phase B: acetonitrile;

[0071] (c) Column temperature: 42±2℃; and / or

[0072] (d) Gradient elution.

[0073] Preferably, the reversed-phase chromatography purification may include the following steps:

[0074] (a) Primary purification: A C8 column was used, with mobile phase A being 40 mmol / L diammonium hydrogen phosphate / water at pH 8.0 and mobile phase B being acetonitrile, using gradient elution; the semaglutide main peak fraction was collected, and the fraction with a purity >97.0% was considered a qualified primary purity fraction; and

[0075] (b) Secondary purification: The qualified fraction of the first purity was purified by gradient elution using a C8 column with 0.1% acetic acid / water as phase A and pure acetonitrile as phase B. The main peak fraction of semaglutide was collected. The fraction with a purity >99.0% and a single impurity <0.1% was considered a qualified fraction of the second purity.

[0076] The main advantages of this invention are:

[0077] 1. Mechanistically, it completely avoids the generation of difficult-to-separate racemic impurities such as Ala at position 30, Ala at position 24, Glu at position 21, Asp at position 15, Thr at position 11, and Glu at position 9, thereby improving product quality and greatly reducing the pressure of subsequent purification and refining, with a purification yield of over 50%.

[0078] 2: While solving key impurities, it retains the convenience of solid-state synthesis process, with low process cost and strong scalability.

[0079] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0080] abbreviation:

[0081] Fmoc 9-fluorenyloxycarbonyl

[0082] Wang Resin

[0083] CTC Resin Chloride Resin

[0084] tBu tert-butyl

[0085] Trt triphenylmethyl

[0086] DCM dichloromethane

[0087] DMF N,N-dimethylformamide

[0088] DMAP 4-Dimethylaminopyridine

[0089] DIEA N,N-Diisopropylethylamine

[0090] DIC N,N-Diisopropylcarbodiimide

[0091] HBTU Benzotriazole-N,N,N',N'-Tetramethylurea hexafluorophosphate

[0092] TBTU O-benzotriazole-N,N,N',N'-tetramethylureatetrafluoroboric acid

[0093] HOBT 1-Hydroxybenzotriazole

[0094] HOAT 1-hydroxy-7-azobenzotriazole

[0095] TFA (trifluoroacetic acid)

[0096] SMT Semaglutide

[0097] The method for detecting racemates is as follows:

[0098] Instrument: Waters Hclass UPLC

[0099]

[0100] The racemic impurities at each point of semaglutide were located by chromatography, and the results are as follows:

[0101] Table 1

[0102]

[0103]

[0104] As shown in Table 1, the impurities generated by the racemization of Asp at position 15, Glu at position 21, Ala at position 24, Glu at position 9, Thr at position 11, and Ala at position 30 are most similar in nature to the main peak and are the most difficult to remove during the purification stage. Therefore, controlling the racemization of these six sites during solid-phase synthesis is of utmost importance.

[0105] Example 1: Synthesis of Fmoc-Gly-Wang Resin

[0106] Weigh 200.00g (Sub=0.50mmol / g) of WangResin and place it in a solid-phase reactor. Wash the resin twice with DMF and swell the resin with DCM for 30 min, 2.0L / time.

[0107] Weigh 89.10 g (300.0 mmol, 3.0 eq) of Fmoc-Gly-OH and 40.50 g (300.0 mmol, 3.0 eq) of HOBt, dissolve them in 1000.0 ml of a 1:1 DMF / DCM mixed solution, pre-freeze to -5 to 5°C, add 48.00 ml (300.0 mmol, 3.0 eq) of DIC and activate for 5 min.

[0108] After the resin swelled and the reaction was completed, the mixture was mechanically stirred and protected with nitrogen. After 3 minutes of reaction, 3.66 g (30 mmol, 0.3 eq) of DMAP was dissolved in 100 ml of DCM and added to the reaction. The reaction temperature was controlled at 25 ± 5 °C and the coupling reaction was carried out for 3.0 h. The reaction solution was removed and the resin was washed 4 times with DMF, 2.0 L each time. A small sample was taken and dried. The Sub was measured to be 0.36 mmol / g, and the synthesis scale was approximately 80 mmol.

[0109] Add 1.0L of acetylation reagent (150.0ml acetic anhydride, 125.0ml pyridine, 725ml DMF) to the resin and acetylate for 60min. Dry the resin under vacuum and wash it 6 times with DMF, 2.0L each time.

[0110] Example 2: Synthesis of Fmoc-Gly-Wang Resin

[0111] Weigh 200.00g (Sub=0.50mmol / g) of Wang Resin and place it in a solid-phase reactor. Wash the resin twice with DMF and swell the resin with DCM for 30 min, 2.0L / time.

[0112] Weigh 89.10 g (300.0 mmol, 3.0 eq) of Fmoc-Gly-OH and 40.50 g (300.0 mmol, 3.0 eq) of HOBt, dissolve them in 1000.0 ml of a 1:1 DMF / DCM mixed solution, pre-freeze to -5 to 5°C, add 48.00 ml (300.0 mmol, 3.0 eq) of DIC and activate for 5 min.

[0113] After the resin swelled and the reaction was completed, the mixture was mechanically stirred and protected with nitrogen. After 3 minutes of reaction, 3.66 g (30 mmol, 0.3 eq) of DMAP was dissolved in 100 ml of DCM and added to the reaction. The reaction temperature was controlled at 25 ± 5 °C and the coupling reaction was carried out for 3.0 h. The reaction solution was removed and the resin was washed 4 times with DMF, 2.0 L each time. A small sample was dried and the Sub was measured to be 0.35 mmol / g. The synthesis scale was approximately 77 mmol. Subsequent additions were made at 80 mmol.

[0114] Add 1.0L of acetylation reagent (150.0ml acetic anhydride, 125.0ml pyridine, 725ml DMF) to the resin and acetylate for 60min. Dry the resin under vacuum and wash it 6 times with DMF, 2.0L each time.

[0115] Example 3: Preparation of Semaglutide Peptide Resin

[0116] The Fmoc-Gly-Wang Resin synthesized in Example 1 was deprotected twice with 20% piperidine / DMF, 2.0 L / time, for 5 min the first time and 25 min the second time; after deprotection, it was washed 6 times with DMF, 2.0 L / time. A small sample was tested with Kaiser's reagent, and the resin turned deep blue.

[0117] Weigh 155.52 g (240 mmol, 3.0 eq) of Fmoc-Arg-OH and 32.40 g (240.0 mmol, 3.0 eq) of HOBt, dissolve them in 1000.0 ml of DMF / DCM solution at a volume ratio of 1:1, pre-freeze to -5 to 5°C, add 38.40 ml (240.0 mmol, 3.0 eq) of DIC and activate for 5 min.

[0118] After the resin was washed and the reaction was completed, the mixture was mechanically stirred and protected with nitrogen. The reaction temperature was 25±5℃, and the coupling reaction was carried out for 2.0 h. The reaction solution was removed, and the resin was washed 6 times with DMF, 2.0 L each time. A small sample was taken and tested with Kaiser reagent. The resin was colorless, indicating that the condensation of Arg at position 36 was completed.

[0119] Following a similar coupling method to that used for the 36-position Arg, and in accordance with the semaglutide peptide sequence, the following are sequentially linked: Fmoc-Gly-OH, Fmoc-Arg-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ala-Trp(Boc)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Ala-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmo c-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Aib-OH, Boc-His(Trt)-OH, Ste-γ-Glu(AEEA-AEEA-OH)-OtBu (side chain) were used to obtain a fully protected semaglutide peptide resin. The resin was washed 6 times with DMF and 3 times with DCM, 2.0 L / wash. The resin was then vacuum dried at 25°C for 8-12 h. The total weight of the SMT peptide resin was 660.12 g.

[0120] Example 4: Preparation of Semaglutide Peptide Resin

[0121] The Fmoc-Gly-Wang Resin synthesized in Example 2 was deprotected twice with 20% piperidine / DMF, 2.0 L / time, for 5 min the first time and 25 min the second time; after deprotection, it was washed 6 times with DMF, 2.0 L / time. A small sample was tested with Kaiser's reagent, and the resin turned deep blue.

[0122] Weigh 155.52 g (240 mmol, 3.0 eq) of Fmoc-Arg-OH and 32.40 g (240.0 mmol, 3.0 eq) of HOBt, dissolve them in 1000.0 ml of DMF / DCM solution with a volume ratio of 1:1, pre-freeze to -5 to 5°C, add 38.40 ml (240.0 mmol, 3.0 eq) of DIC and activate for 5 min.

[0123] After the resin was washed and the reaction was completed, the mixture was mechanically stirred and protected with nitrogen. The reaction temperature was 25±5℃, and the coupling reaction was carried out for 2.0 h. The reaction solution was removed, and the resin was washed 6 times with DMF, 2.0 L each time. A small sample was taken and tested with Kaiser reagent. The resin was colorless, indicating that the condensation of Arg at position 36 was completed.

[0124] Following a similar coupling method to that used for the 36-position Arg, and in accordance with the semaglutide peptide sequence, the following are sequentially linked: Fmoc-Gly-OH, Fmoc-Arg-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Ala-OH, 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. The following compounds were added: Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-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, Ste-γ-Glu(AEEA-AEEA-OH)-OtBu (sidechain), to obtain a fully protected semaglutide peptide resin. The resin was washed 6 times with DMF and 3 times with DCM, 2.0 L / wash. The resin was then vacuum dried at 25°C for 8–12 h, and 655.26 g of SMT peptide resin was obtained.

[0125] Example 5: Preparation of crude semaglutide peptide

[0126] Weigh 500g of the semaglutide peptide resin prepared in Example 3 for later use.

[0127] Prepare 5000 ml of lysis reagent with a volume ratio of TFA:TIS:water = 95:2.5:2.5, and pre-freeze to 0±5℃ for later use.

[0128] Under stirring conditions, peptide resin was added to the lysis agent, and the mixture was stirred at 25°C for 2.0 h. The reaction was then stopped, and the mixture was filtered. The resin was washed twice with TFA (500 ml each time). The filtrates were combined and slowly poured into 36 L of pre-cooled methyl ether. The mixture was stirred and allowed to stand at room temperature for 0.5–1.0 h. The methyl ether precipitate was centrifuged at 3000 rpm for 3 min, and washed / centrifuged three times with methyl ether. The resulting solid was dried to obtain 246.33 g, with a yield of 98.79%. Analysis of the crude peptide sample showed a purity of 70.13%.

[0129] Example 6: Preparation of crude semaglutide peptide

[0130] Weigh 500g of the semaglutide peptide resin prepared in Example 4 for later use.

[0131] Prepare 5000 ml of lysis reagent with a volume ratio of TFA:TIS:water = 95:2.5:2.5, and pre-freeze to 0±5℃ for later use.

[0132] Under stirring conditions, peptide resin was added to the lysis agent, and the mixture was stirred at 25°C for 2.0 h. The reaction was then stopped, and the mixture was filtered. The resin was washed twice with TFA (500 ml each time). The filtrates were combined and slowly poured into 36 L of pre-cooled methyl ether. The mixture was stirred and allowed to stand at room temperature for 0.5–1.0 h. The methyl ether precipitate was centrifuged at 3000 rpm for 3 min, and washed / centrifuged three times with methyl ether. The resulting solid was dried to obtain 238.25 g, with a yield of 98.75%. Analysis of the crude peptide sample showed a purity of 69.54%.

[0133] Example 7: Purification of crude semaglutide peptide

[0134] Weigh 30.0g of the crude peptide obtained in Example 5, dissolve it in 3000ml of ammonia / water at pH 8.0, and filter it through a 0.45um filter membrane.

[0135] On a C8 preparative column with an inner diameter of 150 mm, mobile phase A was 40 mmol / L diammonium hydrogen phosphate / water (pH 8.0), and mobile phase B was pure acetonitrile. The flow rate was 600 mL / min, and the detection wavelength was 210 nm. Gradient elution was performed, and the fraction containing the main peak of semaglutide was collected. Fractions with an analytical purity >97.0% were considered qualified primary pure fractions. The primary pure gradient elution program is as follows:

[0136] B％ 20 35 45 50 60 60

[0137] The first-purity qualified fraction was purified in two steps using a gradient elution process: 0.1% acetic acid / water as phase A and pure acetonitrile as phase B, at a flow rate of 600 ml / min and a detection wavelength of 210 nm. The fraction containing the main peak of semaglutide was collected. The qualified second-purity fraction was defined as having a purity >99.0% and a single impurity <0.1%. The gradient elution procedure for the second-purity fraction is as follows:

[0138] B％ 20 40 51 55 60

[0139] The qualified fraction was subjected to rotary evaporation and then freeze-dried to obtain 15.40g of semaglutide peptide with a purity of 99.60% and a total yield of 51.21%.

[0140] Using the above-described racemic mixture detection method, the results of the protamine detection are as follows:

[0141]

[0142]

[0143] Example 8: Purification of crude semaglutide peptide

[0144] Weigh 30.0g of the crude peptide obtained in Example 6, dissolve it in 3000ml of ammonia / water at pH 8.0, and filter it through a 0.45um filter membrane.

[0145] On a C8 preparative column with an inner diameter of 150 mm, mobile phase A was 40 mmol / L diammonium hydrogen phosphate / water at pH 8.0, and mobile phase B was pure acetonitrile. The flow rate was 600 mL / min, and the detection wavelength was 210 nm. Gradient elution was performed, and the fraction containing the main peak of semaglutide was collected. Fractions with a purity >97.0% were considered pure and qualified.

[0146] The first-purity qualified fraction was purified in two steps using 0.1% acetic acid / water as phase A and pure acetonitrile as phase B, with a flow rate of 600 ml / min and a detection wavelength of 210 nm, by gradient elution. The fraction with the main peak of semaglutide was collected. The fraction with a purity >99.0% and a single impurity <0.1% was considered the second-purity qualified fraction.

[0147] The qualified fraction was subjected to rotary evaporation and then freeze-dried to obtain 10.80g of semaglutide peptide with a purity of 99.32% and a total yield of 36.87%.

[0148] Using the above-described racemic mixture detection method, the results of the protamine detection are as follows:

[0149] <![CDATA[D-Asp 15 -SMT]]> 0.960 0.03 <![CDATA[D-Glu 21 -SMT]]> 0.969 0.05 <![CDATA[D-Ala 24 -SMT]]> 0.982 0.02 Main component 1.000 99.32 <![CDATA[D-Glu 9 -SMT]]> 1.010 0.08 <![CDATA[D-Thr 11 -SMT]]> 1.012 0.05 <![CDATA[D-Ala 30 -SMT]]> 1.014 0.05

[0150] In summary, this invention identifies the most difficult-to-separate impurity combination among the racemic impurities of semaglutide. By using a specific dipeptide feeding method during synthesis, the generation of these difficult-to-separate impurities is avoided, significantly reducing the purification difficulty of crude semaglutide. Simultaneously, it reduces the feeding of other easily separable racemic dipeptides, thus effectively controlling costs. Results demonstrate that the method of this invention produces semaglutide with high yield and high purity, suitable for industrial production.

[0151] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A solid-phase synthesis method for semaglutide, characterized in that, Including the following steps: (a) Using Wang resin as a solid-phase carrier, Fmoc-Gly-OH is condensed in the presence of a coupling agent to obtain the amino acid resin Fmoc-Gly-Resin; then, it is coupled with the corresponding amino acid precursor according to the peptide sequence of semaglutide, and fed in the form of dipeptides at positions 30 (Ala), 24 (Ala), 21 (Glu), 15 (Asp), 11 (Thr), and 9 (Glu) to obtain a fully protected semaglutide peptide resin; the initial substitution degree of Wang resin is 0.4~0.5 mmol / g, the substitution degree of Fmoc-Gly-Resin is 0.35~0.40 mmol / g, and the coupling agent for each coupling reaction is HOBt / DIC; (b) Cleavage the semaglutide resin to obtain crude semaglutide; In step (a), the semaglutide is coupled sequentially according to the following peptide sequence: Fmoc-Arg(pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ala-Trp(Boc)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Ala-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-L eu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(t Bu)-OH, Fmoc-Thr(tBu)-Phe-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Aib-OH, Boc-His(Trt)-OH, and Ste-γ-Glu(AEEA-AEEA-OH)-OtBu (side chain) yields fully protected semaglutide peptide resin, with the following structure: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(side chain)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-Resin; The method further includes the steps of: reversed-phase chromatography purification, salt conversion, and freeze-drying to obtain semaglutide peptide; The reversed-phase chromatography purification includes the following steps: (a) Primary purification: A C8 column was used, with mobile phase A being 40 mmol / L diammonium hydrogen phosphate / water at pH 8.0 and mobile phase B being acetonitrile, using gradient elution; the main peak fraction of semaglutide was collected, and the fraction with a purity >97.0% was considered a qualified primary purification fraction. The primary purification gradient elution procedure is as follows: ； and (b) Secondary purification: The qualified fraction of the first purity was purified by gradient elution using a C8 column with 0.1% acetic acid / water as phase A and pure acetonitrile as phase B. The main peak fraction of semaglutide was collected. The fraction with a purity >99.0% and a single impurity <0.1% was considered a qualified fraction of the second purity. The secondary purification gradient elution procedure is as follows: 。 2. The synthesis method according to claim 1, characterized in that, In step (a), each coupling reaction independently includes one or more of the following characteristics: (1) The amino acid precursor is first de-Fmoc group removed before the reaction, and the de-Fmoc reagent is 20% piperidine / DMF, by volume ratio; (2) The molar ratio of resin to amino acid precursor is 1:2.0-3.

5. The amount of resin used is calculated based on its total degree of substitution. (3) The molar ratio of amino acid precursor to coupling agent is 1:1-1.5; (4) The reaction solvent is a mixed solution of DMF / DCM; and / or (5) DIC or DCC activating coupling agent is also added to the reaction solution.

3. The synthesis method as described in claim 1, characterized in that, In step (a), each coupling reaction independently includes one or more of the following characteristics: (1) The reaction is carried out in an inert gas atmosphere; (2) The reaction temperature is 25±10℃; (3) The reaction time is 1-6 hours; and / or (4) After a reaction is completed, the reaction solution is separated, acetylation reagent is added for acetylation, and the resin is washed to proceed to the next reaction or to end the reaction.

4. The synthesis method according to claim 1, characterized in that, In step (b), the cleavage reagent is a TFA solution containing 5-10% by volume a scavenger, wherein the scavenger is selected from the group consisting of dithiothreitol, anisole, phenol, mercaptoethanol, water, and triisopropylsilane, or combinations thereof.

5. The synthesis method as described in claim 2, characterized in that, The reaction solvent is a mixed solution of DMF / DCM in a volume ratio of 1:

1.

6. A solid-phase synthesis method for semaglutide, characterized in that, Including the following steps: (i) Synthesis of Fmoc-Gly-Wang Resin: Weigh 200.00g of WangResin with a substitution degree of 0.50mmol / g and place it in a solid-phase reactor. Wash the resin twice with DMF and swell the resin with DCM for 30min, 2.0L / time. Weigh 89.10 g of Fmoc-Gly-OH and 40.50 g of HOBt, dissolve them in 1000.0 ml of a 1:1 DMF / DCM mixed solution, pre-freeze to -5~5℃, add 48.00 ml of DIC and activate for 5 min; After the resin swells, the reaction is carried out with mechanical stirring and nitrogen protection. After 3 minutes of reaction, 3.66g of DMAP is dissolved in 100ml of DCM and added to the reaction. The reaction temperature is controlled at 25±5℃ and the coupling reaction is carried out for 3.0 hours. The reaction solution is then removed and the resin is washed with DMF 4 times, 2.0L each time. The resin was acetylated with 1.0 L of acetylation reagent for 60 min, dried under vacuum, and washed 6 times with DMF, 2.0 L each time; wherein the acetylation reagent was 150.0 ml of acetic anhydride, 125.0 ml of pyridine and 725 ml of DMF; (ii) Preparation of semaglutide peptide resin: The Fmoc-Gly-Wang Resin synthesized in step (i) was de-Fmoc twice with 20% piperidine / DMF. 2.0L / cycle, first time 5min, second time 25min; after deprotection, wash 6 times with DMF, 2.0L / cycle; Weigh 155.52g of Fmoc-Arg-OH and 32.40g of HOBt, dissolve them in 1000.0ml of DMF / DCM solution with a volume ratio of 1:1, pre-freeze to -5~5℃, add 38.40ml of DIC and activate for 5min; After the resin was washed, it was fed into the reaction chamber, mechanically stirred and protected with nitrogen. The reaction temperature was 25±5℃, and the coupling reaction was carried out for 2.0 h. The reaction liquid was then removed, and the resin was washed with DMF 6 times, 2.0 L each time. Following a similar coupling method to that used for the 36-position Arg, and in accordance with the semaglutide peptide sequence, the following are sequentially linked: Fmoc-Gly-OH, Fmoc-Arg-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ala-Trp(Boc)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Ala-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmo c-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Aib-OH, Boc-His(Trt)-OH, Ste-γ-Glu(AEEA-AEEA-OH)-OtBu (side chain) were used to obtain fully protected semaglutide peptide resin. The resin was washed 6 times with DMF and 3 times with DCM, 2.0 L / wash. The resin was then vacuum dried at 25°C for 8-12 h and weighed to obtain semaglutide peptide resin. (iii) Preparation of crude semaglutide: Weigh 500 g of the semaglutide peptide resin prepared in step (ii) for later use; Prepare 5000 ml of lysis reagent with a volume ratio of TFA:TIS:water = 95:2.5:2.5, and pre-freeze to 0±5℃ for later use; Under stirring conditions, the peptide resin was added to the lysis agent and stirred at 25°C for 2.0 h. The reaction was then stopped, and the mixture was filtered. The resin was washed twice with TFA, 500 ml each time. The filtrates were combined and slowly poured into 36 L of pre-cooled methyl ether. The mixture was stirred and allowed to stand at room temperature for 0.5–1.0 h. The methyl ether precipitate was centrifuged at 3000 r / min for 3 min each time, and washed / centrifuged three times with methyl ether. The resulting solid was dried to obtain crude semaglutide peptide. (iv) Purification of crude semaglutide: Weigh 30.0 g of crude semaglutide obtained in step (iii), dissolve it in 3000 ml of ammonia / water solution at pH 8.0, and filter it through a 0.45 μm filter membrane; On a C8 preparative column with an inner diameter of 150 mm, mobile phase A was 40 mmol / L diammonium hydrogen phosphate / water at pH 8.0, mobile phase B was pure acetonitrile, the flow rate was 600 ml / min, and the detection wavelength was 210 nm; gradient elution was performed, and the semaglutide main peak fraction was collected. The fraction with a purity >97.0% was considered a pure qualified fraction. A pure gradient elution procedure is as follows: The first-purity qualified fraction was purified in two steps using 0.1% acetic acid / water as phase A and pure acetonitrile as phase B, with a flow rate of 600 ml / min and a detection wavelength of 210 nm, by gradient elution. The fraction with the main peak of semaglutide was collected. The fraction with a purity >99.0% and a single impurity <0.1% was considered the second-purity qualified fraction. The two-stage gradient elution procedure is as follows: The two qualified fractions were rotary evaporated and then freeze-dried to obtain semaglutide peptide.

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