Preparation method of long-acting glucagon peptide analogue

Abstract

The invention provides the preparation method of the polypeptide shown in the formula (I), and the preparation method is stable in process, low in impurity content, few in process steps, short in production period and suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of polypeptide drug preparation, specifically to a method for preparing a long-acting glucagon peptide analog. Background Technology

[0002] Glucagon-like peptide-1 (GLP-1) is a small peptide processed from proglucagon expressed in vivo in specific tissues. GLP-1 can stimulate the proliferation and regeneration of pancreatic β cells and prevent pancreatic β cell apoptosis, and can be used to treat type 2 diabetes and obesity. Compared with insulin, GLP-1 has a lower risk of causing hypoglycemia. Glucose-dependent insulinotropic peptide, also known as gastric inhibitory peptide (GIP), is a physiological intestinal hypoglycemic hormone that can lower blood glucose by stimulating insulin secretion and inhibiting gastric acid secretion. Therefore, several dual-active or even multi-active hybrid peptide drugs are currently being investigated for the treatment of diabetes. For example, WO2022133797A1 (hereinafter referred to as background technology in this invention) discloses a glucagon peptide analog with long-acting hypoglycemic effect, showing good hypoglycemic effect. Among them, Example 4 discloses a polypeptide with code P29F, the structure of which is shown in formula (I):

[0003]

[0004] The publicly disclosed synthesis method is complex, contains many impurities, and is costly, and still needs further optimization. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing the polypeptide according to formula (I) below. The preparation method has stable process, low impurity content, few process steps and short production cycle, and is suitable for large-scale industrial production.

[0006]

[0007] The technical solution adopted in this invention is as follows:

[0008] (1) Starting from the amino resin, amino acids are coupled in a solid phase according to the peptide sequence, up to the 15th site (the sites are counted from the N-terminus of the peptide sequence).

[0009] (2) For the 14th site, selectively protected Fmoc-Lys(alloc)-OH or alloc-Lys(Fmoc)-OH is used for coupling.

[0010] In (3-1), when using Fmoc-Lys(alloc)-OH, the subsequent amino acids are coupled sequentially to Boc-His(Trt)-OH; Alloc on Lys is removed, and Fmoc-AEEA-OH*2, Fmoc-Glu-OtBu, and eicosanoic acid monotert-butyl ester are coupled sequentially, and the crude peptide is obtained after cleavage.

[0011] (3-2) When using alloc-Lys(Fmoc)-OH, first remove the Fmoc protecting group, then sequentially couple Fmoc-AEEA-OH*2, Fmoc-Glu-OtBu, and eicosanoic acid monotert-butyl ester; remove Alloc from Lys and the remaining amino acids to amino acid Boc-His(Trt)-OH or Fmoc-His(Trt)-OH (after coupling, Fmoc protection needs to be removed) and then cleave to obtain the crude peptide.

[0012] In a preferred embodiment of the present invention, the amino resin is selected from amino resins such as Rink Amide MBHA and Rink Amide AM.

[0013] In the preferred embodiment of the present invention, the coupling agent used in steps (1) to (3) is any one of HOBt / DIC, PyBop / DIEA, HBTU / DIEA, and HATU / DIEA, and more preferably HOBt / DIC.

[0014] Preferably, the coupling reaction temperature in step (1) is 20-40°C, more preferably 25-35°C.

[0015] Preferably, the activation reaction temperature in step (2) is 0-15℃, more preferably 5-8℃.

[0016] In a preferred embodiment of the present invention, the deFmoc reagent in the coupling process is a DBLK solution, namely 20% piperidine / DMF (V / V).

[0017] In the preferred embodiment of the present invention, steps (4) and (6) are performed by removing Alloc using zero-valent Pd and a reducing agent.

[0018] In a preferred embodiment of the present invention, the crude peptide is further cleaved using a lysis buffer. The lysis buffer may be a mixture of trifluoroacetic acid, anisole, phenol, triisopropylsilane, ethylenedithiol, dithiothreitol, and water in different proportions; preferably, the lysis buffer is selected from trifluoroacetic acid (85-95)%, anisole (0-10%), phenol (0-10%), triisopropylsilane (0-10%), ethylenedithiol (0-10%), dithiothreitol (0-10%), and water (0-10%).

[0019] Preferably, the pyrolysis temperature is 10–40°C, more preferably 20–30°C. The pyrolysis time is ≥1 hour.

[0020] In a preferred embodiment of the present invention, the crude peptide is further purified by a preparative liquid phase. Preferably, the stationary phase of the preparative liquid phase is selected from any one or a combination of C18 (Daisogel: sp-120-40 / 60-C18-RPS) and C8 (Daisogel: sp-120-10-C8-P).

[0021] Beneficial effects of this invention:

[0022] (1) The preparation method described in this invention reduces the synthesis steps of lysine (W2) with side chain modification by optimizing the coupling sequence and selecting specific sites, thereby reducing material consumption and lowering costs; by selectively protecting Lys, the coupling difficulty of lysine in this step is reduced, thus reducing the synthesis difficulty of the product.

[0023] (2) The preparation method described in this invention can significantly improve the purity of the synthesized polypeptide, reduce the number of impurities from more than 10 to no more than 5, reduce the purification difficulty and steps by more than 50%, and reduce the content of the largest single impurity by at least 2 times.

[0024] (3) The preparation method described in this invention reduces the coupling time of side-chain amino acids compared to the steps recorded in WO2022133797A1, reducing the overall synthesis time by at least 10%, shortening the production cycle, and lowering the cost.

[0025] (4) The materials used in the preparation method described in this invention are reduced by half compared to the steps described in WO2022133797A1, namely [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid, Fmoc-Glu-OtBu and octadecanoic acid monotert-butyl ester. In addition, the purity is increased, and the total material cost is reduced by more than 50%. While ensuring the purity of the peptide, the production cost is further reduced. Detailed Implementation

[0026] 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 that do not specify specific conditions are generally performed under conventional conditions in the art or as recommended by the manufacturer. Unless otherwise specified, the reagents or raw materials used in the present invention are commercially available.

[0027] The main reagents and equipment used in this invention are as follows:

[0028] Rinker amide MBHA resin

[0029] 2-CTC resin

[0030] Freeze dryer (Beijing Boyikang, FD-2A).

[0031] The specific meanings of the abbreviations used in this invention are shown in the table below:

[0032]

[0033]

[0034] Comparative Example 1

[0035] Referring to the scheme described in Example 4 of WO2022133797A1, the prepared polypeptide (I) is as follows:

[0036] W2's composition:

[0037] Weigh 20 g of 2-CTC resin with a degree of substitution of 1.0 mmol / g and add it to a solid-phase reaction column. Wash the column once with DMF and swell the resin with DMF for 30 minutes. Dissolve 8.53 g of Alloc-Lys(Fmoc)-OH (20 mmol) in DMF, activate it with 7.5 ml of DIEA (45 mmol) under an ice-water bath, and add it to the reaction column containing the resin. After reacting for 2 hours, add 30 ml of anhydrous methanol to block for 1 hour and wash three times with DMF. Remove the Fmoc protection with a DMF:pyridine mixed solution of 4:1 (v / v), and wash six times with DMF. Weigh 15.42 g of [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid and 5.41 g of HOBt, dissolve them in DMF, activate them with 6.2 ml of DIC under an ice-water bath, and add them to the reaction column containing the resin. React at room temperature for 2 hours. Repeat the steps of removing Fmoc protection and adding the corresponding materials for coupling, sequentially adding [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid, Fmoc-Glu-OtBu, and tert-butyl octadecanoate according to the branched fragment order. After coupling, the resin was washed three times with DMF and five times with MeOH, and then dried under vacuum. The resin was added to 400 ml of TFEA / DCM = 1:4 and reacted at room temperature for 4 h. After filtering the resin, the DCM was removed from the filtrate by rotary evaporation, and the filtrate was added to 500 ml of MTBE for precipitation. After centrifugation and drying, 20.12 g of the target compound was obtained.

[0038] Synthesis of peptide resins:

[0039] 2.85 g of Rink amide MBHA resin with a substitution degree of 0.35 mmol / g was weighed and added to a solid-phase reaction column. The column was swollen with 20 mL of DCM for 30 minutes, followed by washing three times with 20 mL of DMF each time. After washing, 10 mL of DBLK solution (20% piperidine / DMF (V / V)) was added to the column, and the reaction was allowed to proceed for 5 minutes. The column was then filtered, washed once with 20 mL of DMF, and then 10 mL of DBLK solution (20% piperidine / DMF (V / V)) was added again, and the reaction was allowed to proceed for 10 minutes. The Kaiser assay was positive. The column was then filtered, and washed three times with 20 mL of DMF each time. Separately, Fmoc-Ser(tBu)-OH (1.91 g, 5.0 eq) and HOBt (0.81 g, 6.0 eq) were dissolved in 10 mL of DMF. After activation with DIC (0.69 g, 5.5 eq) at 5-8 °C for 5 min, the solution was added to the reaction column and reacted for 1 hour. The reaction was complete after a Kaiser test showed a negative result. The sample was washed three times with 20 mL of DMF each time. The above deprotection and coupling procedures were repeated, and the coupling of other amino acids was completed sequentially according to the amino acid sequence of the peptide, where X... 14 W2 was used for coupling, and Pd(PPh3)4 was used to remove the Alloc group. After the last amino acid was coupled, the protection was performed according to the above deprotection method. After complete deprotection, the mixture was washed twice with DMF, twice with MeOH, twice with DCM, and twice with MeOH, with 20 mL of solvent each time. The material was collected and dried under reduced pressure at room temperature to obtain the target peptide resin.

[0040] Cleavage of crude peptides:

[0041] Weigh 4.93 g of the above-mentioned peptide resin and slowly add it to 60 mL of lysis buffer (trifluoroacetic acid: anisole: anisole: ethylene dithiol = 90:5:3:2) at 20-30℃. React for 2 hours after addition. After the reaction is complete, filter to remove the resin. With vigorous stirring, pour the filtrate into pre-cooled methyl ether (600 mL). Allow the resulting mixture to settle in a refrigerator for 2 hours. Remove the supernatant and wash five times with pre-cooled methyl ether (400 mL each time). Collect the material and dry under reduced pressure at room temperature to obtain 2.37 g of crude peptide.

[0042] Purification of crude peptides:

[0043] The crude peptide was purified using a preparative liquid chromatography system (Beijing Innovation Tongheng, LC3000) through multiple steps: Step 1: Stationary phase: C18 (Daisogel: sp-120-40 / 60-C18-RPS), mobile phase: 0.1% TFA, acetonitrile; Step 2: Stationary phase: C8 (Daisogel: sp-120-10-C8-P), mobile phase: 0.5% phosphoric acid, acetonitrile; Step 3: Stationary phase: C8 (Daisogel: sp-120-10-C8-P), mobile phase: 50mM ammonium acetate, 0.3% acetic acid, acetonitrile. Finally, the purified peptide was obtained by lyophilization.

[0044] MS test result m / z 4778.5963(M+H)+.

[0045] Example 1

[0046] Synthesis of peptide resins:

[0047] 2.85 g of Rink amide MBHA resin with a substitution degree of 0.35 mmol / g was weighed and added to a solid-phase reaction column. The column was swollen with 20 mL of DCM for 30 minutes, followed by washing three times with 20 mL of DMF each time. After washing, 10 mL of DBLK solution (20% piperidine / DMF (V / V)) was added to the column, and the reaction was allowed to proceed for 5 minutes. The column was then filtered, washed once with 20 mL of DMF, and then 10 mL of DBLK solution (20% piperidine / DMF (V / V)) was added again, and the reaction was allowed to proceed for 10 minutes. The Kaiser assay was positive. The column was then filtered, and washed three times with 20 mL of DMF each time. Separately dissolve Fmoc-Ser(tBu)-OH (1.91 g, 5.0 eq) and HOBt (0.81 g, 6.0 eq) in 10 mL of DMF. Activate the solution with DIC (0.69 g, 5.5 eq) at 5-8 °C for 5 min, then add it to the reaction column. React for 1 hour. Kaiser test is negative, indicating the reaction is complete. Wash the solution 3 times with 20 mL of DMF each time. Repeat the above deprotection and coupling operations, sequentially coupling other amino acids according to the peptide's amino acid sequence, coupling to the modification site (site 14). Using Fmoc-Lys(Alloc)-OH with selective side-chain protection, remove the Fmoc protecting group from the side chain, and sequentially couple the subsequent amino acids to Boc-His(Trt)-OH. Remove the Alloc from Lys, and sequentially couple Fmoc-AEEA-OH*2, Fmoc-Glu-OtBu, and eicosanoic acid monotert-butyl ester. After coupling, wash twice with DMF, twice with MeOH, twice with DCM, and twice with MeOH, each time using 20 mL of solvent. Collect the material and dry under reduced pressure at room temperature to obtain the target peptide resin.

[0048] Cleavage of crude peptides:

[0049] Weigh 4.88 g of the above-mentioned peptide resin and slowly add it to 60 mL of lysis buffer (trifluoroacetic acid: anisole: anisole: ethylene dithiol = 90:5:3:2) at 20-30℃. React for 2 hours after addition. After the reaction is complete, filter to remove the resin. With vigorous stirring, pour the filtrate into pre-cooled methyl ether (600 mL). Allow the resulting mixture to settle in a refrigerator for 2 hours. Remove the supernatant and wash five times with pre-cooled methyl ether (400 mL each time). Collect the material and dry under reduced pressure at room temperature to obtain 2.23 g of crude peptide.

[0050] Purification of crude peptides:

[0051] The crude peptide was purified using a preparative liquid chromatography system (Beijing Innovation Tongheng, LC3000) through multiple steps: Step 1: Stationary phase: C18 (Daisogel: sp-120-40 / 60-C18-RPS), mobile phase: 0.1% TFA, acetonitrile; Step 2: Stationary phase: C8 (Daisogel: sp-120-10-C8-P), mobile phase: 0.5% phosphoric acid, acetonitrile; Step 3: Stationary phase: C8 (Daisogel: sp-120-10-C8-P), mobile phase: 50mM ammonium acetate, 0.3% acetic acid, acetonitrile. Finally, the purified peptide was obtained by lyophilization.

[0052] Example 2

[0053] Synthesis of peptide resins:

[0054] 2.85 g of Rink amide MBHA resin with a substitution degree of 0.35 mmol / g was weighed and added to a solid-phase reaction column. The column was swollen with 20 mL of DCM for 30 minutes, followed by washing three times with 20 mL of DMF each time. After washing, 10 mL of DBLK solution (20% piperidine / DMF (V / V)) was added to the column, and the reaction was allowed to proceed for 5 minutes. The column was then filtered, washed once with 20 mL of DMF, and then 10 mL of DBLK solution (20% piperidine / DMF (V / V)) was added again, and the reaction was allowed to proceed for 10 minutes. The Kaiser assay was positive. The column was then filtered, and washed three times with 20 mL of DMF each time. Separately, Fmoc-Ser(tBu)-OH (1.91 g, 5.0 eq) and HOBt (0.81 g, 6.0 eq) were dissolved in 10 mL of DMF. After activation with DIC (0.69 g, 5.5 eq) at 5-8 °C for 5 min, the solution was added to the reaction column and reacted for 1 hour. The reaction was complete, indicated by a negative Kaiser assay. The sample was washed three times with 20 mL of DMF each time. The above deprotection and coupling procedures were repeated, and other amino acids were coupled sequentially according to the peptide's amino acid sequence, up to the modification site (site 14). The side-chain-selectively protected Alloc-Lys(Fmoc)-OH was used to remove the Fmoc protecting group, followed by coupling with Fmoc-AEEA-OH*2, Fmoc-Glu-OtBu, and tert-butyl eicosanoate. After coupling the side chains, zero-valent Pd and a reducing agent were used to remove the Alloc from Lys, and the remaining amino acids on the main chain were coupled. After the last amino acid, Boc-His(Trt)-OH, was coupled, the sample was washed twice with DMF, twice with MeOH, twice with DCM, and twice with MeOH, with 20 mL of solvent used for each wash. The sample was collected and dried under reduced pressure at room temperature to obtain the target peptide resin.

[0055] Cleavage of crude peptides:

[0056] Weigh 5.10 g of the above-mentioned peptide resin and slowly add it to 60 mL of lysis buffer (trifluoroacetic acid: anisole: anisole: ethylene dithiol = 90:5:3:2) at 20-30℃. React for 2 hours after addition. After the reaction is complete, filter to remove the resin. With vigorous stirring, pour the filtrate into pre-cooled methyl ether (600 mL). Allow the resulting mixture to settle in a refrigerator for 2 hours. Remove the supernatant and wash five times with pre-cooled methyl ether (400 mL each time). Collect the material and dry under reduced pressure at room temperature to obtain 2.52 g of crude peptide.

[0057] Purification of crude peptides:

[0058] The crude peptide was purified using a preparative liquid chromatography system (Beijing Innovation Tongheng, LC3000) through multiple steps: Step 1: Stationary phase: C18 (Daisogel: sp-120-40 / 60-C18-RPS), mobile phase: 0.1% TFA, acetonitrile; Step 2: Stationary phase: C8 (Daisogel: sp-120-10-C8-P), mobile phase: 0.5% phosphoric acid, acetonitrile; Step 3: Stationary phase: C8 (Daisogel: sp-120-10-C8-P), mobile phase: 50mM ammonium acetate, 0.3% acetic acid, acetonitrile. Finally, the purified peptide was obtained by lyophilization.

[0059] Example 3: Purity determination of synthesized polypeptides

[0060] Determination method: Instrument: Analytical high performance liquid chromatograph; Column packing: octadecylsilane-bonded silica gel; Mobile phase: Phase A: 10 mmol / L diammonium hydrogen phosphate solution, Phase B: acetonitrile; Column temperature: 40℃; Flow rate: 0.8 ml / min; Detection wavelength: 220 nm; Elution gradient as follows:

[0061]

[0062] The measurement results are as follows:

[0063]

[0064] Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for preparing a polypeptide of formula (I), wherein the method comprises the following steps: (1) coupling amino acids according to the peptide sequence starting from an amino resin, to the 15th position (the position is counted from the N-terminal of the peptide sequence), (2) coupling Fmoc-Lys(alloc)-OH or alloc-Lys(Fmoc)-OH with selective protection at the 14th position, (3-1) when Fmoc-Lys(alloc)-OH is used, sequentially coupling the following amino acids to Boc-His(Trt)-OH; removing Alloc on Lys, sequentially coupling Fmoc-AEEA-OH*2, Fmoc-Glu-OtBu, eicosanedioic acid mono-tert-butyl ester, and obtaining a crude peptide after cleavage; (3-2) when alloc-Lys(Fmoc)-OH is used, sequentially coupling Fmoc-AEEA-OH*2, Fmoc-Glu-OtBu, eicosanedioic acid mono-tert-butyl ester after removing the Fmoc protecting group; removing Alloc on Lys and the remaining amino acids to couple with amino acid Boc-His(Trt)-OH or Fmoc-His(Trt)-OH (Fmoc protection needs to be removed after coupling is completed), and obtaining a crude peptide after cleavage. The amino resin is selected from Rink Amide MBHA and Rink Amide AM; the coupling agent used in steps (1)-(3) is any one of HOBt / DIC, PyBop / DIEA, HBTU / DIEA, and HATU / DIEA, and more preferably HOBt / DIC. The coupling reaction temperature in step (1) is 20-40°C, and more preferably 25-35°C; preferably, the activation reaction temperature in step (2) is 0-15°C, and more preferably 5-8°C. wherein The Fmoc removal reagent in the coupling process is a DBLK solution, i.e., 20% piperidine / DMF (V / V); preferably, the Alloc in steps (4) and (6) is removed by using zero-valent Pd and a reducing agent. The crude peptide is further cleaved with a cleavage solution; preferably, the cleavage solution is prepared by mixing trifluoroacetic acid, benzyl mercaptan, anisole, phenol, triisopropylsilane, ethanedithiol, dithiothreitol, and water in different proportions; preferably, the cleavage solution is selected from trifluoroacetic acid (85-95)%, benzyl mercaptan (0-10%), anisole (0-10%), phenol (0-10%), triisopropylsilane (0-10%), ethanedithiol (0-10%), dithiothreitol (0-10%), and water (0-10%).

2. The production process according to claim 1, characterized in that The cleavage temperature is 10-40°C, and more preferably 20-30°C. The cleavage time is ≥1 hour.

3. The production process according to claim 1 or 2, characterized in that, The crude peptide is further purified by a preparation liquid phase; preferably, the stationary phase of the preparation liquid phase is selected from any one of C18 (Daisogel: sp-120-40 / 60-C18-RPS) and C8 (Daisogel: sp-120-10-C8-P), or a combination thereof.

4. The production process according to claims 1 to 3, characterized in that ​ 5. The production method according to claim 4, characterized by, ​ 6. The method of claim 1, wherein the method is characterized by, ​ 7. The production process according to claims 1 to 6, characterized in that ​

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