Semaglutide intermediate and preparation method thereof, and synthesis method of Semaglutide

Abstract

The present invention relates to the field of medical technology, and in particular to a semaglutide intermediate and a preparation method thereof, and a method for synthesizing semaglutide. The present invention screens different solvent combinations, finds a reaction system compatible with side chain tetrapeptide modification, main chain dipeptide modification and deprotection steps, and synthesizes semaglutide using a "one-pot method". The method does not require separation of any intermediate, does not involve complex operations such as isoelectric elution, separation, and redissolution of the intermediate, is simple to operate, has a high yield, and is conducive to industrial scale-up production. Further, the present invention uses a double Fmoc-protected dipeptide: Fmoc-His(Fmoc)-Aib-OH replaces Fmoc-His-Aib-OH·TFA as a starting material, which greatly reduces the use of Fmoc-His(Fmoc)-Aib-OH, and the feed amount is reduced from 8 to 10 times to 1 to 3 times, the atomic economy is increased, and the production cost is reduced.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a semaglutide intermediate and a preparation method thereof, and a synthesis method of semaglutide. Background Art

[0002] Semaglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist developed by Nordisk of Denmark. It was approved by the U.S. Food and Drug Administration (FDA) in December 2017 for blood sugar control in adults with type 2 diabetes. In addition, in June 2021, Semaglutide was also approved by the FDA for weight loss, becoming the first GLP-1 receptor agonist for weight management.

[0003] Since semaglutide is obtained by chemically modifying the human GLP-1 structure, it contains non-natural amino acids in its side chains and main chains and cannot be directly expressed by biological recombination. The preparation process selected by Novo Nordisk is to produce the GLP-1 (11-37) fragment by yeast fermentation, and then obtain semaglutide by subsequent chemical modification.

[0004] In the original process of Novo Nordisk for synthesizing semaglutide [CN110041399A], after Arg34GLP-1 (9-37) is produced by yeast fermentation, semaglutide is obtained by subsequent chemical modification. In the back-end chemical modification part, Arg34-GLP-1 (9-37) is first acylated with Ste-Glu (AEEA-AEEA-OSu) -OH to obtain an acylated intermediate, but since the reaction system at this time is an aqueous phase, it is incompatible with the next step of coupling the Fmoc-His-Aib-OH·TFA dipeptide system. Therefore, the method adopted by Novo Nordisk is to perform complex operations such as isoelectric precipitation, centrifugation, and dissolving the acylated intermediate precipitate with NMP before proceeding to the next step of reaction. Not only is the process complicated, but the cycle is long and the efficiency is low. Summary of the invention

[0005] In view of this, the present invention provides a semaglutide intermediate and a preparation method thereof, and a method for synthesizing semaglutide.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] Semaglutide dipeptide intermediate, its structural formula is as follows:

[0008]

[0009] in,

[0010] R1 and R2 are the same or different amino protecting groups, and

[0011] R1 and R2 are independently selected from Boc, Trt, Bpoc, Fmoc, Cbz, Alloc, ivDde or Nps.

[0012] In a specific embodiment of the present invention, the semaglutide dipeptide intermediate is a double Fmoc-protected dipeptide: Fmoc-His(Fmoc)-Aib-OH, and its structural formula is as follows:

[0013]

[0014] The present invention also provides a method for preparing Fmoc-His(Fmoc)-Aib-OH, comprising:

[0015] Fmoc-His-Aib-OH·TFA reacts with Fmoc reagent to obtain Fmoc-His(Fmoc)-Aib-OH.

[0016] The Fmoc reagent includes Fmoc-OSu or Fmoc-Cl.

[0017] The present invention also provides the use of the semaglutide dipeptide intermediate (Fmoc-His(Fmoc)-Aib-OH) in the synthesis of semaglutide.

[0018] The present invention also provides a method for preparing semaglutide, which is characterized by comprising the following steps:

[0019] S1, reacting the Ste-Glu(AEEA-AEEA-OSu)-OH fragment with Arg34-GLP-1(9-37) in a solvent to obtain the M1 intermediate;

[0020] S2, activating the semaglutide dipeptide intermediate and reacting it with the M1 intermediate to obtain the M2 intermediate;

[0021] S3, deprotecting the M2 intermediate to obtain semaglutide;

[0022] The semaglutide dipeptide intermediate is the above-mentioned double Fmoc-protected dipeptide: Fmoc-His(Fmoc)-Aib-OH.

[0023] The method for preparing semaglutide of the present invention screens different solvent combinations and finds a reaction system that is compatible with side chain tetrapeptide modification (i.e., step S1), main chain dipeptide modification (i.e., step S2) and deprotection step (i.e., step S3): one or more of MeCN, THF, DMF, DMAc, NMP and DMSO mixed with water in any proportion. The subsequent chemical modification of the Arg34-GLP-1 (9-37) fragment and the subsequent semaglutide synthesis reaction are completed by a "one-pot method". The method does not require separation of any intermediates and does not involve complex operations such as electroelution, separation, and re-dissolution of intermediates in CN110041399A. The yield is significantly improved, and the operation is simpler, which is conducive to industrial scale-up.

[0024] In the above-mentioned "one-pot" reaction system of the present invention, in step S2, when the semaglutide dipeptide intermediate is Fmoc-His-Aib-OH·TFA, generally, the higher the proportion of water, the higher the reaction rate and the more thorough the reaction, but it will result in a very large feed ratio of Fmoc-His-Aib-OH·TFA during the subsequent coupling with Fmoc-His-Aib-OH·TFA, and the production cost is still not ideal.

[0025] On this basis, the present invention further discovered that the use of a double Fmoc-protected dipeptide: Fmoc-His(Fmoc)-Aib-OH, instead of Fmoc-His-Aib-OH·TFA for feeding can greatly reduce the amount of feed, thereby reducing production costs. The structural formula of Fmoc-His(Fmoc)-Aib-OH is as follows:

[0026]

[0027] Experiments show that when Fmoc-His(Fmoc)-Aib-OH is used instead of Fmoc-His-Aib-OH·TFA as feed, the hydrolysis rate of Fmoc-His(Fmoc)-Aib-OH is significantly slowed down after being activated and added to the reaction system, thereby greatly reducing the usage of Fmoc-His(Fmoc)-Aib-OH, thereby increasing atom economy and significantly reducing production costs.

[0028] Arg34-GLP-1 (9-37) can be a common commercial product or can be prepared by itself. In a specific embodiment of the present invention, Arg34-GLP-1 (9-37) is commercially available and purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0029] In the present invention, in step S1, the solvent is composed of water and at least one of the following components in any proportion: MeCN, THF, DMF, DMAc, NMP, DMSO. In a specific embodiment of the present invention, the solvent is composed of water and NMP, wherein the volume ratio of NMP to water is 2:1.

[0030] In the present invention, in step S2, the activation reagent used for the activation includes triethylamine / DIPEA, A and B; the A is HOBt or HOAt, and the B is PyBOP, PyAOP, HATU, HBTU, TBTU, DCC, DIC or EDCI.

[0031] In the present invention, in step S2, the pH of the reaction is 8.0-10.0, specifically 8.0, 8.5, 9.0, 9.5, 10.0.

[0032] In some embodiments of the present invention, in step S2, the semaglutide dipeptide intermediate is Fmoc-His(Fmoc)-Aib-OH, and its feeding amount relative to Arg34-GLP-1(9~37) is 1.0~3.0 equivalents, specifically 1.0 equivalent, 1.5 equivalents, 2.0 equivalents, 2.5 equivalents, and 3.0 equivalents.

[0033] In the present invention, in step S3, the deprotection reagent is piperidine. In a specific embodiment of the present invention, the deprotection reagent is a piperidine solution with a volume fraction of 5%.

[0034] The present invention screens different solvent combinations, discovers a reaction system that is compatible with side chain tetrapeptide modification, main chain dipeptide modification and deprotection steps, and uses a "one-pot method" to synthesize semaglutide. The method does not require separation of any intermediates, does not involve complex operations such as isoelectric elution, separation, and re-dissolution of intermediates, is simple to operate, has a high yield, and is conducive to industrial scale-up production. Furthermore, the present invention uses a double Fmoc-protected dipeptide: Fmoc-His (Fmoc) -Aib-OH to replace Fmoc-His-Aib-OH·TFA as a starting material, which greatly reduces the use of Fmoc-His (Fmoc) -Aib-OH, and the feed amount is reduced from 8 to 10 times to 1 to 3 times, the atom economy is significantly improved, and the production cost is greatly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The synthetic route map of semaglutide;

[0036] Figure 2 is the hydrogen spectrum of Fmoc-His(Fmoc)-Aib-OH dipeptide;

[0037] Figure 3is the carbon spectrum of Fmoc-His(Fmoc)-Aib-OH dipeptide;

[0038] Figure 4 is the mass spectrum of Fmoc-His(Fmoc)-Aib-OH dipeptide;

[0039] Figure 5 is the high performance liquid chromatogram of Fmoc-His(Fmoc)-Aib-OH dipeptide;

[0040] Figure 6 This is the liquid chromatogram of semaglutide;

[0041] Figure 7 This is the high-resolution mass spectrum of semaglutide. DETAILED DESCRIPTION

[0042] The present invention provides a method for preparing a semaglutide intermediate and its application in the synthesis of semaglutide. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0043] The synthetic route of semaglutide provided by the present invention is as follows Figure 1 As shown, the specific steps include:

[0044] S1. An acylation coupling reaction is carried out between Ste-Glu(AEEA-AEEA-OSu)-OH and the amino group on the 26-lys of Arg34-GLP-1(9-37) in a solvent at a pH of 10 to 12 to obtain an M1 intermediate reaction solution having the structure: H-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Ste)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH;

[0045] S2, subjecting the Fmoc-His(Fmoc)-Aib-OH dipeptide to an acylation coupling reaction with the N-terminal amino group in the M1 intermediate under the action of a condensation activation reagent to obtain an M2 intermediate reaction solution, wherein the structure of the M2 intermediate is:

[0046] Fmoc-His(Fmoc)-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Ste)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH;

[0047] The S3 and M2 intermediate reaction solutions are subjected to the action of piperidine to remove the Fmoc protecting group to obtain a crude semaglutide solution.

[0048] In step S1, the entire "one-pot" reaction system is selected from one or more of MeCN, THF, DMF, DMAc, NMP and DMSO and mixed with water in any proportion.

[0049] In step S2, the condensation activation reagent is triethylamine / DIPEA+A+B, wherein A is HOBt or HOAt, and B is one of PyBOP, PyAOP, HATU, HBTU, TBTU, DCC, DIC, and EDCI.

[0050] In step S2, the pH of the acylation coupling reaction is between 8.0 and 10.0.

[0051] In step S2, the feeding amount of Fmoc-His(Fmoc)-Aib-OH is between 1.0 and 3.0 equivalents.

[0052] In the present invention, the preparation method further comprises step S4: purifying the crude semaglutide solution to obtain refined semaglutide. The present invention does not make special requirements on the specific purification method, and any purification method for polypeptide compounds commonly used in the art can be used.

[0053] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0054] Abbreviations and English meanings

[0055] Abbreviations and English and Chinese meanings MeCN Acetonitrile DMAc N,N-Dimethylacetamide DMF N,N-Dimethylformamide NMP N-Methylpyrrolidone DMSO Dimethyl sulfoxide DIPEA N,N-Diisopropylethylamine HPLC High Performance Liquid Chromatography HOBt 1-Hydroxybenzotriazole HATU 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate HBTU Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate DIPEA N,N-Diisopropylethylamine PyBOP Benzotriazol-1-yl-oxytripyrrolidino hexafluorophosphate PyAOP (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tris-1-pyrrolidinylphosphonium hexafluorophosphate. TBTU O-Benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate

[0056] The present invention will be further described below in conjunction with embodiments:

[0057] Example 1: Synthesis of Fmoc-His(Fmoc)-Aib-OH

[0058] Weigh Fmoc-His-Aib-OH·TFA (57.7 g, 100.0 mmol) and Fmoc-OSu (40.4 g, 120 mmol, 1.2 eq.) into a 2 L round-bottom flask, add 1.0 L of DMF, stir to dissolve, then weigh DIPEA (25.8 g, 200 mmol, 2.0 eq.) and add dropwise to the reaction solution. After the addition is complete, react at room temperature for 2 to 5 h (TLC detection). After the reaction is completed, add 2.0 L of 15% citric acid aqueous solution to acidify the reaction solution, then add 2.0 L of ethyl acetate for extraction, collect the organic phase, wash the organic phase twice with 2.0 L of 15% citric acid aqueous solution and 2.0 L of 25% sodium chloride water, respectively, and then dry with an appropriate amount of anhydrous sodium sulfate for 1 h, filter, and collect the organic phase. Finally, the organic phase was added dropwise to 6.0 L of n-heptane to precipitate a large amount of solids. The mixture was stirred overnight and filtered. The filter cake was washed three times with an appropriate amount of n-heptane and dried to obtain 62.5 g of Fmoc-His(Fmoc)-Aib-OH with a yield of 91.4%. Its hydrogen spectrum, carbon spectrum, mass spectrum and liquid chromatography are shown in the following table. Figures 2 to 5 shown.

[0059] NMR data: 1HNMR(600MHz,DMSO)δ12.42(s,1H),8.16(s,1H),8.12–8.05(m,1H),7.94–7.85(m,4H) ,7.77–7.59(m,4H),7.51(dd,J=34.6,8.6Hz,1H),7.46–7.37(m,4H),7.37–7.31(m,3H ),7.30–7.25(m,1H),4.67(dd,J=6.7,2.5Hz,2H),4.49–4.32(m,2H),4.28–4.15(m,3H ),2.95–2.86(m,1H),2.85–2.77(m,1H),1.38(d,J=4.9Hz,3H),1.33(d,J=12.4Hz,3H); 13 C NMR(151MHz,DMSO)δ175.89,170.95,156.17,148.52,144.24,144.18,143.49,141.23 ,141.15,141.11,140.48,139.89,137.90,137.00,135.02,129.40,128.40,128.08,1 27.78,127.77,127.48,127.46,125.76,125.71,125.59,121.86,120.73,120.55,120.51,114.69,110.24,69.69,66.15,55.46,54.10,47.06,46.52,31.73,25.32,25.18.

[0060] Example 2: One-pot synthesis of semaglutide using Fmoc-His(Fmoc)-Aib-OH dipeptide

[0061] Arg34-GLP-1(9-37) (17.5 g, 5.0 mmol, content 91.0%) was weighed, 800 mL of NMP:H2O (2:1) was added, and then 1.0 M NaOH aqueous solution was added and stirred to dissolve Arg34-GLP-1(9-37) and control the pH value of the peptide solution between 10.5 and 11.5. Ste-Glu(AEEA-AEEA-OSu)-OH (6.7 g, 7.5 mmol, content 93.6%, 1.5 eq.) was weighed, 10 times NMP was added until dissolved by stirring, and then injected into the peptide solution at a constant speed by injection pump. During the injection process, the pH value of the reaction solution was controlled to be stable between 10.5 and 11.5 by adding 0.5 M NaOH aqueous solution. After the injection was completed, the pH value of the reaction solution was adjusted to between 8.5 and 9.5 with 15% citric acid to obtain the M1 intermediate reaction solution. Fmoc-His(Fmoc)-Aib-OH (6.8 g, 10.0 mmol, 2.0 eq.), HOBt (1.3 g, 11.0 mmol, 2.2 eq.) and PyBOP (5.2 g, 10.0 mmol, 2.0 eq.) were weighed and added to the reaction bottle, 50 ml NMP was added and stirred, and then DIPEA (5.2 g, 40.0 mmol, 8.0 eq.) was weighed and added to the reaction solution for activation. After activation for 30 min, it was added dropwise to the peptide solution. After the addition was completed, the reaction was continued for 2 to 5 h to obtain the M2 intermediate reaction solution. After the reaction was completed, 5% piperidine was added to the total reaction volume and the reaction was continued for 1.0 h. After the reaction was completed, it was filtered and the filtrate was adjusted to a pH value between 7.5 and 8.0 with 15% citric acid to obtain a crude semaglutide solution. After purification and freeze-drying by reverse phase preparative HPLC, 14.5 g of semaglutide was obtained, with a total yield of 70.5%. Figure 6 , high resolution mass spectrum see Figure 7 .

[0062] Comparative Example 1: Synthesis of semaglutide by “one-pot method” using Fmoc-His-Aib-OH·TFA dipeptide

[0063] Arg34-GLP-1(9-37) (17.5 g, 5.0 mmol, content 91.0%) was weighed, 800 mL of NMP:H2O (2:1) was added, and then 1.0 M NaOH aqueous solution was added and stirred to dissolve Arg34-GLP-1(9-37) and control the pH value of the peptide solution between 10.5 and 11.5. Ste-Glu(AEEA-AEEA-OSu)-OH (6.7 g, 7.5 mmol, content 93.6%, 1.5 eq.) was weighed, 10 times NMP was added until dissolved by stirring, and then injected into the peptide solution at a constant speed by injection pump. During the injection process, the pH value of the reaction solution was controlled to be stable between 10.5 and 11.5 by adding 0.5 M NaOH aqueous solution. After the injection was completed, the pH value of the reaction solution was adjusted to between 8.5 and 9.5 with 15% citric acid to obtain the M1 intermediate reaction solution. Fmoc-His-Aib-OH·TFA (24.2 g, 42.0 mmol, 8.4 eq.), HOBt (5.9 g, 44.0 mmol, 8.8 eq.) and PyBOP (20.8 g, 40.0 mmol, 8.0 eq.) were weighed and added to the reaction bottle, 200 ml of NMP was added and stirred, and then DIPEA (20.6 g, 160.0 mmol, 32 eq.) was weighed and added to the reaction solution for activation. After activation for 30 min, it was added to the peptide solution at a uniform speed using a syringe pump. During the injection process, the pH value of the reaction solution was adjusted to be stable between 8.5 and 9.5 with 0.5 M NaOH / 10% phosphoric acid. After the addition was completed, the reaction was continued for 2 to 5 h to obtain the M2 intermediate reaction solution. After the reaction, 5% piperidine was added to the total reaction volume and the reaction was carried out for 1.0 h. After the reaction was completed, the solution was filtered and the pH value of the filtrate was adjusted to between 7.5 and 8.0 with 15% citric acid to obtain a crude semaglutide solution. After purification and freeze-drying by reverse phase preparative HPLC, 13.8 g of semaglutide was obtained, and the total yield was 67.1%.

[0064] By comparing the dipeptide feed amount and yield of Example 2 and Comparative Example 1, it can be seen that the dipeptide protected by double Fomc in the present application can significantly reduce the use of dipeptide equivalents in the synthesis process of semaglutide, and the feed amount is reduced from 8 to 10 times to 1 to 3 times, the atom economy is significantly improved, and the production cost is greatly reduced.

[0065] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Semaglutide dipeptide intermediate, the structural formula of which is as follows: in, R1 and R2 are the same or different amino protecting groups, and R1 and R2 are independently selected from Boc, Trt, Bpoc, Fmoc, Cbz, Alloc, ivDde or Nps.

2. The dipeptide intermediate according to claim 1, characterized in that: The R1 and R2 are both Fmoc, and their structural formula is as follows:

3. The method for preparing the semaglutide dipeptide intermediate according to claim 2, characterized in that: include: Fmoc-His-Aib-OH·TFA reacts with Fmoc reagent to obtain Fmoc-His(Fmoc)-Aib-OH; The Fmoc reagent includes Fmoc-OSu or Fmoc-Cl.

4. Use of the semaglutide intermediate according to claim 1 or 2 or the semaglutide dipeptide intermediate obtained according to claim 3 in the synthesis of semaglutide.

5. A method for preparing semaglutide, characterized in that: The steps include: S1, reacting the Ste-Glu(AEEA-AEEA-OSu)-OH fragment with Arg34-GLP-1(9-37) in a solvent to obtain the M1 intermediate; S2, activating the semaglutide dipeptide intermediate and reacting it with the M1 intermediate to obtain the M2 intermediate; S3, deprotecting the M2 intermediate to obtain the crude semaglutide peptide; The semaglutide dipeptide intermediate is the semaglutide dipeptide intermediate according to any one of claims 1 to 2 or the semaglutide dipeptide intermediate prepared according to claim 3.

6. The preparation method according to claim 5, characterized in that: In step S1, the solvent is composed of water and at least one of the following components in any proportion: Composition: MeCN, THF, DMF, DMAc, NMP, DMSO; preferably, the solvent consists of water and NMP, and the volume ratio of NMP to water is 2:

1.

7. The preparation method according to claim 5, characterized in that: In step S2, the activation reagent used in the condensation includes triethylamine / DIPEA, A and B; A is HOBt or HOAt, and B is PyBOP, PyAOP, HATU, HBTU, TBTU, DCC, DIC or EDCI.

8. The preparation method according to claim 5, characterized in that: In step S2, the pH of the reaction is 8.0-10.

0.

9. The preparation method according to claim 5, characterized in that: In step S2, the feeding amount of the semaglutide dipeptide intermediate is 1.0 to 3.0 equivalents.

10. The preparation method according to any one of claims 5 to 9, characterized in that: The method also includes a step of purifying the crude semaglutide peptide.

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