Preparation method of semeglutide

Abstract

The invention relates to the technical field of polypeptide drugs, in particular to a preparation method of semeglutide. The preparation method comprises the following steps: sequentially coupling the following peptide fragments to synthesize the full-protection peptide resin of the semeglutide; the peptide fragments 7-10 are Boc-His (Trt)-Aib-Glu (OtBu)-Gly-OH, and the peptide fragments 7-10 are Boc-His (Trt)-Aib-Glu (OtBu)-Gly-OH; the peptide fragment 11 to the peptide fragment 12 are Fmoc-Thr (tBu)-Phe-OH; the peptide fragment 13 to the peptide fragment 16 are Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-Val-OH; the peptide fragments 17 to 18 are Fmoc-Ser (tBu)-Ser (tBu)-OH; the peptide fragment 19-22 is Fmoc-Tyr (tBu)-Leu-Glu (OtBu)-Gly-OH, and the peptide fragment 19-22 is a peptide fragment 19-22; the peptide fragment 23 to the peptide fragment 25 are Fmoc-Gln (Trt)-Ala-Ala-OH; and the peptide fragment 35 to 36 is Fmoc-Gly-Arg (Pbf)-OH. The preparation method provided by the invention has the advantages of few steps, high yield and high product purity.

Description

Technical Field

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

[0002] Semaglutide, a GLP-1 analogue with 94% homology to human GLP-1, is a long-acting glucagon-like 1 (GLP-1) analog developed and manufactured by Novo Nordisk, Denmark, for the treatment of type 2 diabetes. In December 2017, semaglutide injection was approved for marketing in the United States. In September 2019, the oral formulation of semaglutide was approved by the FDA for glycemic control in type 2 diabetes. In June 2021, the injectable formulation was approved by the FDA for weight loss management in overweight adults. Furthermore, research has found that semaglutide has the potential to treat cancer, Alzheimer's disease, and Parkinson's disease, and to reduce the risk of cardiovascular disease.

[0003] Its structural formula is as follows Figure 1 As shown, structurally, Semaglutide is formed by replacing 8 Aib positions on the GLP-1 (7-37) chain.

[0004] In Ala, Arg replaces Lys at position 34, and an octadecanoic acid fatty chain is attached to Lys at position 26, along with glutamic acid and a short-chain PEG modification. PEG modification not only allows it to bind tightly to albumin, masking the DPP-4 enzyme hydrolysis site, but also reduces renal excretion, prolonging the biological half-life and achieving a long-lasting effect.

[0005] Currently, the most efficient chemical synthesis method for peptide drugs is solid-phase synthesis. This involves starting from the carboxyl terminus of the peptide, attaching the first amino acid to a resin, removing the protecting group from the amino group, and then coupling the next amino acid according to the peptide sequence, removing the protecting group, and repeating this cycle until amino acid assembly is complete. After cleaving the peptide from the resin and removing all protecting groups, the crude peptide is obtained. This crude peptide is then purified and lyophilized to obtain the active pharmaceutical ingredient (API).

[0006] Synthesizing smegglutide using conventional solid-phase methods presents significant challenges, primarily due to the presence of numerous hydrophobic amino acids in the main chain, which stabilize the hydrogen bonds between peptide chains, leading to severe folding; enhanced intermolecular forces causing resin shrinkage and increasing the difficulty of amino acid coupling; reduced coupling reactivity and efficiency, resulting in lower reaction yields and increased costs. Therefore, the industrial-scale chemical preparation of smegglutide faces considerable difficulties.

[0007] CN116120427A discloses a method for synthesizing smegglutinin, but the total yield of the crude smegglutinin synthesized by this method is only 57.07%, and the HPLC purity of the crude smegglutinin is only 77.71%.

[0008] Therefore, there is an urgent need to establish a low-cost, high-quality, and simple method for the industrial-scale preparation of smegglutinin. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a method for preparing smegglutinin with high yield and high purity.

[0010] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0011] A method for preparing smegglutinin includes the step of using a solid-phase synthesis method to sequentially couple an amino acid, a dipeptide fragment, a tripeptide fragment, and a tetrapeptide fragment from the C-terminus to the N-terminus of smegglutinin according to its primary sequence to obtain a fully protected peptide resin of smegglutinin.

[0012] The dipeptide fragments include at least two of peptides 11-12, 17-18, and 35-36.

[0013] The peptide segments 11-12 are Fmoc-Thr(tBu)-Phe-OH;

[0014] Peptide segments 17-18 are Fmoc-Ser(tBu)-Ser(tBu)-OH;

[0015] Peptide segments 35-36 are Fmoc-Gly-Arg(Pbf)-OH;

[0016] The tripeptide fragment is peptide 23-25;

[0017] The peptide segments 23-25 ​​are Fmoc-Gln(Trt)-Ala-Ala-OH;

[0018] The tetrapeptide fragment is at least two of peptides 7-10, 13-16, and 19-22.

[0019] The peptides 7-10 are Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH;

[0020] Peptide segments 13-16 are Fmoc-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-OH;

[0021] The peptides 19-22 are Fmoc-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH;

[0022] The fully protected peptide resin sequence of smegglutinin is as follows:

[0023] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-S er(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEE A]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin.

[0024] In one embodiment, the dipeptide fragment includes peptides 11-12, 17-18, and 35-36.

[0025] The peptide segments 11-12 are Fmoc-Thr(tBu)-Phe-OH;

[0026] Peptide segments 17-18 are Fmoc-Ser(tBu)-Ser(tBu)-OH;

[0027] Peptide segments 35-36 are Fmoc-Gly-Arg(Pbf)-OH;

[0028] The tripeptide fragment is peptide 23-25;

[0029] The peptide segments 23-25 ​​are Fmoc-Gln(Trt)-Ala-Ala-OH;

[0030] The tetrapeptide fragments are peptides 7-10, 13-16, and 19-22.

[0031] The peptides 7-10 are Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH;

[0032] Peptide segments 13-16 are Fmoc-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-OH;

[0033] The peptides 19-22 are Fmoc-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH;

[0034] When dipeptide, tripeptide, or tetrapeptide fragments are not used, a stepwise synthesis method can be used.

[0035] In general, solid-phase synthesis differs from liquid-phase synthesis in that each step of a solid-phase synthesis reaction cannot achieve complete coupling. Therefore, the more condensation steps are performed in solid-phase synthesis, the greater the decrease in yield. Thus, in this embodiment, by using pre-synthesized dipeptide, tripeptide, and tetrapeptide fragments for coupling, the number of condensation reactions can be significantly reduced, thereby significantly improving the reaction yield.

[0036] Understandably, in this embodiment, the amino acid has the necessary side-chain protecting groups to avoid unwanted side reactions, such as Fmoc, Pbf, OtBu, tBu, and Boc.

[0037] In one embodiment, the solid-phase synthesis carrier of the smegglutinin fully protected peptide resin is Wang resin.

[0038] In one embodiment, the degree of substitution of the Wang resin is 0.20 to 0.83 mmol / g, preferably 0.45 to 0.50 mmol / g.

[0039] In one embodiment, the preparation method includes the step of sequentially coupling the amino acid and / or the dipeptide fragment to obtain a first resin conjugate, wherein the first resin conjugate is...

[0040] Fmoc-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin.

[0041] In an optional embodiment, the first resin coupling compound is obtained by sequentially coupling Fmoc-Gly-OH, Fmoc-Gly-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH·H2O, Fmoc-Ile-OH, Fmoc-Phe-OH, and Fmoc-G1u(OtBu)-OH·H2O.

[0042] In one embodiment, during the synthesis of the precursor Fmoc-Gly-Arg(Pbf)-Gly-Wang Resin of the first resin conjugate, the side-chain protecting group Fmoc, which is attached to the residue Arg(Pbf), is removed using a deprotecting agent containing piperidine.

[0043] In one embodiment, the preparation method further includes the step of removing the side chain protecting group Fmoc from the first resin coupling compound;

[0044] Then, Fmoc-L-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-OH is coupled to obtain the second resin conjugate;

[0045] The second resin coupling compound is

[0046] Fmoc-L-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Ar g(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin.

[0047] In one embodiment, the deprotecting agent for removing the side-chain protecting group Fmoc is a solution of piperidine in N,N-dimethylformamide, and the concentration (V / V) of the deprotecting agent is 20% to 50%, preferably 20%.

[0048] The step of sequentially coupling the dipeptide, tripeptide and tetrapeptide fragments to obtain the smegglutinin fully protected peptide resin;

[0049] The process includes pyrolysis, crystallization, filtration, and drying of the smegglutinin fully protected peptide resin to obtain crude smegglutinin.

[0050] In one alternative embodiment, after the second resin conjugate is deprotected by the peptide chain protecting group Fmoc, it is sequentially coupled with...

[0051] The fully protected peptide resin of smegglutinin was obtained by mixing Fmoc-Gln(Trt)-Ala-Ala-OH, Fmoc-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH, Fmoc-Ser(tBu)-Ser(tBu)-OH, Fmoc-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-OH, Fmoc-Thr(tBu)-Phe-OH, and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH.

[0052] The fully protected peptide resin sequence of smegglutinin is as follows:

[0053] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-S er(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEE A]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin.

[0054] A semaglutide prepared by the aforementioned preparation method, wherein the semaglutide has an HPLC purity of ≥78% and a total yield of ≥82%.

[0055] In this invention, if there is a conflict between the Chinese name and the structural formula of a compound, the structural formula shall prevail, unless the structural formula is obviously incorrect.

[0056] The beneficial effects of this invention are as follows:

[0057] 1. The smegglutinin prepared by the method provided by the present invention has high purity, with a total yield of 82.8% for crude smegglutinin and an HPLC purity of 81.8% for crude smegglutinin.

[0058] 2. The preparation method provided by this invention uses Wang resin as a solid-phase synthesis carrier, which is very suitable for the assembly of peptides with difficult sequences. The prepared products have higher purity and higher coupling efficiency, thereby significantly reducing production costs. Attached Figure Description

[0059] Figure 1 The structural formula of smegglutinin;

[0060] Figure 2 This is a synthetic route diagram for smegglutinin;

[0061] Figure 3 HPLC chromatogram of a purified sample of smegglutinin;

[0062] Figure 4 HPLC chromatograms of two purified semaglutide samples;

[0063] Figure 5 The HPLC chromatogram of the smegglutinin peptide sample;

[0064] Figure 6 This is the HPLC chromatogram of crude smegglutinin. Detailed Implementation

[0065] The present invention is illustrated below with reference to examples, but is not intended to limit the invention. Any simple substitutions or modifications made to the present invention by those skilled in the art are within the scope of the technical solutions protected by this invention.

[0066] In this article, the abbreviations and their Chinese definitions are shown in Table 1.

[0067] Table 1

[0068]

[0069]

[0070] Example

[0071] 1. Synthesis of Fmoc-Gly-Wang Resin

[0072] Weigh 10g of Wang Resin (0.50 mmol / g, purchased from Tianjin Nankai Hecheng Technology Co., Ltd., product number: HCW01-1-1), add it to the reaction vessel, add DMF (125 mL, 12.5V), stir and wash for 5 min, then filter; add...

[0073] DMF (85 mL, 8.5 V) was stirred and swollen for 30 min, then filtered. Fmoc-Gly-OH (4.471 g, 3 eq) and HOBt (2.450 g, 3 eq) were weighed and dissolved in DMF (50 mL, 5 V), stirred for 2 min, poured into a reactor, and DIC (2.80 mL, 3 eq) was added to the reactor. DMAP (184 mg, 0.3 eq) in DMF (5 mL) solution was added, and the mixture was stirred at room temperature for 15 h. The mixture was filtered, and DMF (70 mL, 7 V) was added, stirred for 2 min, for a total of 7 times. Small samples were dried and the degree of substitution of the amino acid resin was measured; the degree of substitution was 0.350–0.420 mmol / g.

[0074] 2. Synthesis of Fmoc-Gly-Arg(Pbf)-Gly-Wang resin

[0075] Add 20% PIP / DMF (80 mL, 8V) to the reactor, stir for 10 min, and filter; add 20% PIP / DMF (80 mL, 8V) to the reactor, stir for 20 min; (IPC: ninhydrin / phenol / pyridine: blue), filter; wash with DMF (80 mL, 8V), repeat 8-12 times, 2 min each time; weigh Fmoc-Gly-Arg ( Pbf)-OH (7.062 g, 2.0 eq) and HOBt (1.638 g, 2.4 eq) were dissolved in pre-cooled DMF (60 mL, 6 V) and poured into a reaction vessel. DIC (1.86 mL, 2.4 eq) was added dropwise and stirred at room temperature for 1-2 h. (IPC: ninhydrin / phenol / pyridine: pale yellow) The mixture was filtered and washed with DMF (80 mL, 8 V) four times, 2 min each time.

[0076] 3. Synthesis of Fmoc-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin

[0077] Add 20% PIP / DMF (80 mL, 8V) to the reactor, stir for 10 min, and filter; add 20% PIP / DMF (80 mL, 8V) to the reactor, stir for 20 min; (IPC: ninhydrin / phenol / pyridine: blue), filter; wash with DMF (80 mL, 8V), repeat 8-12 times, 2 min each time; weigh Fmoc-Arg(Pb) f)-OH (6.530 g, 2.0 eq) and HOBt (1.630 g, 2.4 eq) were dissolved in pre-cooled DMF (60 mL, 6 V) and poured into a reaction vessel. DIC (1.86 mL, 2.4 eq) was added dropwise and stirred at room temperature for 1-2 h. (IPC: ninhydrin / phenol / pyridine: pale yellow) The mixture was filtered and washed with DMF (80 mL, 8 V) 4 times, 2 min each time.

[0078] 4. Synthesis of Fmoc-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin

[0079] Add 20% PIP / DMF (80 mL, 8V) to the reactor, stir for 10 min, and filter. Add 20% PIP / DMF (80 mL, 8V) to the reactor, stir for 20 min (IPC: ninhydrin / phenol / pyridine: blue), and filter. Wash with DMF (80 mL, 8V), repeat 8-12 times, 2 min each time. Weigh Fmoc-Val-OH (3.390 g, 2.0 eq) and HOBt (1.630 g, 2.4 eq), dissolve in pre-cooled DMF (60 mL, 6V), pour into the reactor, add DIC (1.86 mL, 2.4 eq) dropwise, and stir at room temperature for 1-2 h (IPC: ninhydrin / phenol / pyridine: pale yellow), and filter. Wash with DMF (80 mL, 8V), repeat 4 times, 2 min each time.

[0080] 5. The amino acid content of the smegglutinin was sequentially coupled according to the primary amino acid sequence, and the amount of amino acid to be fed was as shown in Table 2 below.

[0081] Table 2

[0082]

[0083] 6. The weight of the obtained fully protected peptide resin of smeglucopyrikin was 39.1g, and the weight gain rate of the peptide resin was 98%.

[0084] 7. Prepare the lysis buffer with a volume ratio of TFA:TIS:BDMT = 90:10:5% of the resin weight, and place it at 0-5℃. Add the lysis buffer of peptide resin 8V to the reactor under a nitrogen flow, gradually adding the peptide resin while stirring and controlling the temperature not to exceed 15℃. Stir at 20-25℃ for 2 hours. Filter out the resin and cool the filtrate to 0-5℃. Add MTBE pre-cooled to -10-5℃ to the filtrate while stirring and controlling the temperature not to exceed 20℃. Filter, wash the solid 4-5 times with an equal volume of MBTE, and vacuum dry at room temperature to obtain 17.0g of crude smegglutinin (purity see [link]). Figure 6 The yield was 82.8%. The crude smegglutinin solution obtained in the examples was purified using an NP7000 SERIALS PUMP Hanbon system with a 50×250 mm C18 column, 8 μm. A first purification was performed using a 0.1% trifluoroacetic acid / acetonitrile mobile phase, and the target peak fraction (purity 98.95%, yield 80%) was collected. Figure 3 The target peak fraction from the first purification was subjected to a second purification and salt exchange using 10 mM NaHCO3. The target peak fraction was collected (purity 99.91%, yield 90%, see...). Figure 4), 12.24 g of smegglutinin was obtained by freeze-drying. The freeze-drying conditions were as follows: the smegglutinin stock solution was filtered through a 0.22 μm filter cartridge, and the filtrate was transferred to a freeze-drying pan (the solution height was controlled at 0.7–1.2 cm). Freeze-drying was carried out according to the parameters in Table 3:

[0085] Table 3

[0086]

[0087]

[0088] After freeze-drying, the vacuum is broken with nitrogen. The finished product must be packaged under a nitrogen atmosphere with a relative humidity of less than 15% RH. The HPLC purity is 99.93%, and the total purified yield is 72%. The HPLC chromatogram of smegglutinin is shown below. Figure 5 .

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A process for the preparation of semaglutide, characterized in that, The step of obtaining the full-protected peptide resin of semaglutide according to a solid-phase synthesis method, sequentially coupling amino acids, dipeptide fragments, tripeptide fragments and tetrapeptide fragments from the C-terminal to the N-terminal of the primary sequence of semaglutide, wherein the dipeptide fragments include at least two of the peptide segment 11-12, the peptide segment 17-18 and the peptide segment 35-36; The peptide segment 11-12 is Fmoc-Thr(tBu)-Phe-OH. The peptide segment 17-18 is Fmoc-Ser(tBu)-Ser(tBu)-OH. The peptide segment 35-36 is Fmoc-Gly-Arg(Pbf)-OH. The tripeptide fragment is the peptide segment 23-25. The peptide segment 23-25 is Fmoc-Gln(Trt)-Ala-Ala-OH. The tetrapeptide fragment is at least two of the peptide segment 7-10, the peptide segment 13-16 and the peptide segment 19-22. The peptide segment 7-10 is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH. The peptide segment 13-16 is Fmoc-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-OH. The peptide segment 19-22 is Fmoc-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH. The sequence of the full-protected peptide resin of semaglutide is: 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[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin. The solid-phase synthesis carrier of the full-protected peptide resin of semaglutide is Wang resin.

2. The production method according to claim 1, wherein The degree of substitution of the Wang resin is 0.20-0.83 mmol / g.

3. The production method according to claim 2, wherein The step of sequentially coupling the amino acids and / or the dipeptide fragments to obtain a first resin conjugate Fmoc-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin.

4. The production method according to claim 2, wherein In the process of synthesizing the precursor Fmoc-Gly-Arg(Pbf)-Gly-Wang Resin of the first resin conjugate, the side chain protection group Fmoc connected to the residue Arg(Pbf) is removed by using a deprotection agent containing piperidine.

5. The production method according to claim 4, wherein ​ 6. The production method according to claim 4, wherein The method further comprises the steps of removing the side chain protection group Fmoc of the first resin conjugate; and coupling Fmoc-L-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-OH to obtain a second resin conjugate; the second resin conjugate is Fmoc-L-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin.

7. The production method according to claim 6, wherein The deprotection agent for removing the side chain protection group Fmoc is a solution of piperidine in N,N-dimethylformamide.

8. The production method according to claim 7, wherein The concentration of the deprotection agent is 20% to 50%.

9. The production method according to claim 6, wherein The method further comprises the steps of removing the side chain protection group Fmoc of the second resin conjugate; sequentially coupling the dipeptide, tripeptide and tetrapeptide fragments to obtain the full-protected peptide resin of semaglutide; and cleaving, crystallizing, filtering and drying the full-protected peptide resin of semaglutide to obtain the crude semaglutide.

10. The production method according to claim 4, wherein The first resin conjugate is obtained by sequentially coupling Fmoc-Gly-OH, Fmoc-Gly-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH·H2O, Fmoc-Ile-OH, Fmoc-Phe-OH and Fmoc-Glu(OtBu)-OH·H2O.

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