Smeglutide

Abstract

The invention provides semeglutide, which is synthesized by adopting the following method, and the specific steps are as follows: 1) segmenting the main chain of semeglutide into three polypeptide fragments: 7-14 sites, 15-22 sites and 23-37 sites; (2) respectively preparing the 7-14-site fragments, the 15-22-site fragments and the 23-37-site fragments through solid-phase synthesis; 3) connecting the 7-14-site fragment with the 15-22-site fragment to form a 7-22-site fragment, and activating the C end of the fragment into active ester; (4) selectively removing an Alloc protecting group of Lys26 in the 23-37-site fragment, and then carrying out fatty acid modification to obtain a modified 23-37-site fragment; 5) in a liquid phase system, coupling the activated 7-22-site fragment and the modified 23-37-site fragment according to a molar ratio of 1: (0.6-1.4) to form a complete polypeptide chain; and 6) removing the protecting group and purifying to obtain a semaglutide finished product. The method provided by the invention can effectively realize high-purity, high-yield and low-cost synthesis of the semeglutide bulk drug.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical intermediate synthesis, and particularly relates to semaglutide. Background Art

[0002] Semaglutide is a glucagon-like peptide 1 (GLP-1) receptor agonist, mainly used for blood glucose control in patients with type 2 diabetes and can also reduce cardiovascular risks. It stimulates insulin secretion, suppresses appetite, and delays gastric emptying by mimicking the action of the GLP-1 hormone. Semaglutide is available in two dosage forms: an injection once a week and an oral tablet once a day.

[0003] The polypeptide sequence of semaglutide is as follows:

[0004] H-His 7 -Aib 8 -Glu 9 -Gly 10 -Thr 11 -Phe 12 -Thr 13 -Ser 14 -Asp 15 -Val 16 -Ser 17 -Ser 18 -Tyr 19 -Leu 20 -Glu 21 -Gly 22 -Gln 23 -Ala 24 -Ala 25 -Lys 26 (AEEA-AEEA-γ-Glu-Octadecanedioic Acid)-Glu 27 -Phe 28 -Ile 29 -Ala 30 -Trp 31 -Leu 32 -Val 33 -Arg 34 -Gly 35 -Arg 36 -Gly 37 -OH.

[0005] Patent WO2009083549A1 prepares semaglutide by using a gene recombination-biological fermentation tandem chemical synthesis process. Its process uses Fmoc-His-Aib-OSu and Lys 26The main chain 29 peptide with side chain connected is reacted to obtain Fmoc-semaglutide, and then the Fmoc is removed by piperidine to obtain crude semaglutide. However, the product produced by this method contains 12-17% disubstituted impurities (33 peptides), namely His-Aib-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. Since the Fmoc of the main chain is partially removed, disubstituted impurities are formed, and this impurity causes the overall yield to be too low.

[0006] CN116120427A discloses a method for synthesizing semaglutide, which uses amino acids or peptide segments to be coupled sequentially from the C-terminus to the N-terminus. The total yield of the crude semaglutide synthesized therefrom is only 57.07%. The HPLC purity of the crude semaglutide is only 77.71%.

[0007] The synthesis method of semaglutide disclosed in CN103848910A is to couple Gly and resin to obtain Gly-resin, and then gradually couple amino acids or amino acid derivatives to obtain the main chain peptide segment and then remove Lys 26 The side chain was connected after the protective group was added. Due to the presence of many impurities, the purification yield of the final product was only 54.05% and the total yield was 14.92%.

[0008] At present, the most effective chemical synthesis method for peptide drugs is solid phase synthesis. The conventional solid phase synthesis method of semaglutide is very challenging, mainly because there are a large number of hydrophobic amino acids in the main chain, which makes the hydrogen bonds between peptide chains stable and can cause serious folding; the interaction between peptide chains is enhanced, causing the resin to shrink, increasing the difficulty of amino acid coupling; reducing the reaction activity and efficiency of coupling, reducing the reaction yield and increasing the cost. The industrial chemical preparation of semaglutide is quite difficult. Summary of the invention

[0009] The object of the present invention is to provide a semaglutide, which can effectively realize the synthesis of semaglutide raw materials with high purity, high yield and low cost.

[0010] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0011] A semaglutide is synthesized by the following method, wherein the specific steps are as follows:

[0012] 1) Divide the semaglutide main chain into three polypeptide fragments: positions 7 - 14, 15 - 22, and 23 - 37;

[0013] 2) Prepare the fragment at positions 7 - 14, the fragment at positions 15 - 22, and the fragment at positions 23 - 37 respectively by solid-phase synthesis;

[0014] 3) Connect the fragment at positions 7 - 14 and the fragment at positions 15 - 22 to form a fragment at positions 7 - 22, and activate the C-terminus of this fragment into an active ester;

[0015] 4) Selectively remove the Alloc protecting group of Lys 26 in the fragment at positions 23 - 37 and then perform fatty acid modification to obtain the modified fragment at positions 23 - 37;

[0016] 5) In a liquid phase system, couple the activated fragment at positions 7 - 22 and the modified fragment at positions 23 - 37 in a molar ratio of 1:0.6 - 1.4 to form a complete polypeptide chain;

[0017] 6) Remove the protecting groups and purify to obtain the finished product of semaglutide.

[0018] Further, the division of the polypeptide fragments in step (1) is specifically as follows:

[0019] The fragment sequence at positions 7 - 14 contains:

[0020] His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu);

[0021] The fragment sequence at positions 15 - 22 contains:

[0022] Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly;

[0023] The fragment sequence at positions 23 - 37 contains:

[0024] Gln(Trt)-Ala-Ala-Lys(Alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly.

[0025] Furthermore, in step (2), the solid-phase synthesis uses Wang resin with a substitution degree of 0.1 - 0.6 mmol / g as the carrier, and in the coupling reaction, the condensing agent is selected from at least one of DIC / HOBT, DIC / HOAT, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, and HATU / HOAT / DIPEA.

[0026] Furthermore, the conditions for selectively removing the Alloc protecting group in step (4) are as follows: using 0.05 - 0.5 equivalents of Pd(PPh3)4 and 5 - 50 equivalents of phenylsilane or morpholine, and the reaction time is 10 - 120 minutes.

[0027] Furthermore, the C-terminal activation in step (3) is achieved by the following method:

[0028] Dissolve the 7 - 22 fragment in dichloromethane or DMF, add N-hydroxysuccinimide (NHS) and the coupling reagent DCC or EDC, and react at 10 - 60 °C for 0.5 - 24 hours to form the NHS ester.

[0029] Furthermore, after the coupling reaction in step (5) is completed, the reagent for cleaving the protecting group is a TFA solution containing 1 - 10% scavengers, and the scavengers are selected from at least two of anisole, benzyl mercaptan, ethylene dithiol, mercaptoethanol, phenol, water, and triisopropylsilane (TIS).

[0030] Furthermore, 85 - 95% TFA, 1 - 5% benzyl mercaptan, 1 - 10% water, 1 - 5% TIS.

[0031] Furthermore, the deprotecting reagent for the solid-phase synthesis in step (2) is a 15 - 25% piperidine / DMF solution by volume, and the deprotection time is 1 - 30 minutes.

[0032] Furthermore, the connection between the 7 - 14 fragment and the 15 - 22 fragment in step (3) is achieved by the following method: reacting the resin of the 15 - 22 fragment with the free carboxylic acid 7 - 14 fragment after removing the Fmoc protection in the presence of a condensing agent, and the amount of the condensing agent used is 2 - 10 times the molar amount of the fragment.

[0033] Furthermore, in step (6), the purification uses high-performance liquid chromatography, the mobile phase is an acetonitrile / water mixed solution, the volume ratio of acetonitrile is 10 - 90%, and the purification temperature is 10 - 40 °C.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The main chain of the present invention is divided into three shorter polypeptide fragments at positions 7-14, 15-22, and 23-37 and synthesized simultaneously. The 7-14 peptide segment and the 15-22 peptide segment are assembled into a 7-22 peptide segment. On the other hand, after the 23-37 peptide segment is assembled, the protecting group A11oc on Lys 26 is selectively removed. In a liquid phase system, the side chain of Lys 26 is fatty acylated, and then the 7-22 peptide segment and the 23-37 peptide segment are ligated in the liquid phase reaction system. After separation and purification, the protecting groups are removed to obtain a crude product. Then, through separation and purification, the finished product is obtained. This method can not only make full use of the high efficiency and easy purification characteristics of the solid-phase synthesis method, but also break up the fragment with a large number of hydrophobic amino acids between His 7 and Lys 26 into smaller parts to avoid the formation of severe β-sheets and resin polycondensation. On the other hand, a liquid phase reaction system is used for the coupling of the 7-22 peptide segment and the 23-37 peptide segment, and the fatty acylation of the side chain of Lys 26 in peptide chain fragment 2. In the liquid phase system, both reaction substrates participate in the reaction at a molar ratio close to 1:1, which has a significant economic advantage compared with the traditional solid-phase synthesis method (usually 1:3-8). BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0037] Figure 1 It is a flow chart of the synthesis method of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] In the following, only some exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the embodiments of the present invention. Therefore, the drawings and the description are considered to be exemplary in nature rather than restrictive.

[0039] In the description of the embodiments of the present invention, it should be understood that the orientation or positional relationship indicated by the terms "length", "vertical", "horizontal", "top", "bottom", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the embodiments of the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the embodiments of the present invention.

[0040] In addition, the terms "first" and "second" are for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the embodiments of the present invention, "a plurality of" means two or more, unless otherwise specifically defined.

[0041] In the embodiments of the present invention, unless otherwise clearly specified and limited, terms such as "mounted", "connected", "coupled", "fixed", etc. should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or integrated; it may be a mechanical connection, an electrical connection, or a communication connection; it may be directly connected, or indirectly connected through an intermediate medium, and may be the internal connection or the interaction relationship between two components. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of the present invention can be understood according to specific circumstances.

[0042] In the embodiments of the present invention, unless otherwise clearly specified and limited, the first feature being "on" or "under" the second feature may include the first and second features being in direct contact, or may include the first and second features not being in direct contact but being in contact through additional features therebetween. Moreover, the first feature being "above", "over" and "on top of" the second feature includes the first feature being directly above and obliquely above the second feature, or merely indicating that the first feature has a higher horizontal height than the second feature. The first feature being "under", "beneath" and "underneath" the second feature includes the first feature being directly below and obliquely below the second feature, or merely indicating that the first feature has a lower horizontal height than the second feature.

[0043] The following disclosure provides many different embodiments or examples for implementing different structures of the embodiments of the present invention. To simplify the disclosure of the embodiments of the present invention, the components and settings of specific examples are described below. Of course, they are merely examples and are not intended to limit the embodiments of the present invention. In addition, the embodiments of the present invention may repeat reference numerals and / or reference letters in different examples. This repetition is for the purpose of simplification and clarity and does not itself indicate the relationship between the various embodiments and / or settings discussed.

[0044] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0045] See Figure 1 , the present invention proposes to solve the above problems by means of fragment synthesis. That is, the main chain is decomposed into 2 key fragments:

[0046] Fragment 1 (7-22 peptide segment):

[0047] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH;

[0048] Fragment 2 (23-37 peptide segment):

[0049] Fmoc-Gln(Trt)-Ala-Ala-Lys(alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH;

[0050] Fragment 3:

[0051] Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic Acid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

[0052] Among them, Fragment 1 can be further decomposed into 2 sub-fragments:

[0053] Fragment 4 (7-14 peptide segment):

[0054] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH;

[0055] Fragment 5 (15-22 peptide segment):

[0056] Fmoc-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH.

[0057] Specifically as follows:

[0058] a. Use Wang resin as the solid-phase carrier. Fragment 2 and Fragment 5 are coupled with Fmoc-G1y-OH to prepare Fmoc-Gly-Wang resin; Fragment 4 is coupled with Fmoc-Ser(tBu)-OH to prepare Fmoc-Ser(tBu)-Wang. Then, the protected amino acids are sequentially coupled and linked according to the sequence to obtain the target peptide segments Fragment 2, Fragment 4 and Fragment 5 respectively.

[0059] b. Cut fragment 4 from the resin and then combine it with fragment 5 that is attached to the resin and has had the Fmoc protection removed:

[0060] (Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Wang) to form fragment 1 attached to the resin: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Wang. Then cut fragment 1 from the resin to form fragment 1: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH.

[0061] c. React the C-terminus of fragment 1 with an activating group such as NHS. Form fragment 1 with an activated C-terminus:

[0062] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu.

[0063] d. Fragment 2 attached to the resin:

[0064] The protecting group Alloc on [[Fmoc-Gln(Trt)-Ala-Ala-Lys(alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang]] is removed to form [[Fmoc-Gln(Trt)-Ala-Ala-Lys-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH]], which is then cleaved from the resin. Subsequently, fatty acylation is carried out in the liquid phase to obtain [[Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH]]. After deprotection at the N-terminus, purification is performed to obtain Fragment 3:

[0065] Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH;

[0066] e. In the liquid phase, the C-terminally activated Fragment 1 is ligated with the peptide prepared in step d to obtain:

[0067] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH-Gln(Trt)-Ala-Ala

[0068] -Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-OH.

[0069] f. The peptide prepared in step e is purified, deprotected and then purified again to obtain the finished product of semaglutide.

[0070] Among them,

[0071] In step a, the solid support is Wang resin with a substitution degree of 0.1 - 0.6 mmol / g. After connecting the amino acid to the resin, after removing the Fmoc protection, the corresponding Fmoc-protected amino acid is added in a feeding ratio of 2 - 5 times for the coupling reaction. Each coupling reaction is a solid-phase peptide coupling reaction carried out in the presence of a condensing agent. The end point of each step of the coupling reaction is detected with Kaiser reagent. After the reaction is completed, the Fmoc is removed with a deprotection reagent, and then coupled with the next Fmoc-protected amino acid; the operation is repeated until the fully protected fragments 2, fragment 4, and fragment 5 are synthesized respectively. The deprotection reagent is preferably a 20% piperidine in DMF solution (volume ratio); the condensing agent used in the coupling reaction is one of the following combinations: DIC / HOBT, DIC / HOAT, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, HATU / HOAT / DIPEA.

[0072] In step b, the method for cleaving the peptide chain from the resin is: reacting the peptide chain attached to the resin with a mixture of TFA, triisopropylsilane (TIPS), and water, with a preferred ratio of 90% TFA, 5% TIPS, and 5% water. The mixture is stirred at room temperature for 2 - 3 hours, a large amount of ether (5 - 50 times the mass of the resin, preferably 15 times) is added to precipitate the crude peptide, and then centrifuged and washed with ether 2 - 4 times to obtain it.

[0073] In step c, the C-terminal exposed peptide segment is dissolved in a dry organic solvent (such as dichloromethane or DMF). NHS is added to the solution, and then a coupling reagent such as DCC or EDC is added. The reaction is stirred at room temperature or slightly higher temperature (usually about 10 - 60 °C, preferably 25 °C) for 0.5 - 20 hours to form the NHS ester intermediate.

[0074] In step d, Lys 26 The deprotection method of the protecting group A11oc on it is to use Pd(PPh3)4 with a dosage of 0.1 - 0.4 times the synthesis scale and phenylsilane (or morpholine) with a dosage of 10 - 30 times the synthesis scale for 10 - 90 minutes.

[0075] In steps b, c, d, and f, the condensing agent used in the coupling reaction is one of the following combinations: DIC / HOBT, DIC / HOAT, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, HATU / HOAT / DIPEA.

[0076] In step e, the cleavage reagent is a TFA solution containing 1-5% scavenger by volume, and the scavenger is one or more of anisole, benzyl methyl sulfide, ethylene dithiol, mercaptoethanol, phenol, water, and TIS. The preferred ratio of the cleavage reagent is: TFA / anisole / water / TIS = 90 / 2.5 / 5.0 / 2.5.

[0077] The abbreviations used in the specification and claims have the following meanings:

[0078]

[0079]

[0080] Specific preparation examples are as follows:

[0081] Example 1: Synthesis of Fmoc-Gly-Wang;

[0082] Place 500.0 g of Wang resin (sub = 0.42 mmol / g) in a synthesis column, wash it twice with 2400 mL of DMF, and swell it with 2400 mL of DCM for 30 min; after filtering off the DCM by suction, add a mixed DCM solution of Fmoc-Gly-OH / DIC / HOBT [weigh 282.4 g (400 mmol) of Fmoc-Gly-OH and 64.8 g (480 mmol) of HOBT into an amino acid activation bottle, add a mixed solution of DMF and DCM with a volume ratio of 1:1 and stir to dissolve, add 76.4 mL (480 mmol) of DIC at 0 °C, activate for 5 minutes, after reacting for 10 min, add 4.8 g (4 mmol) of DMAP: react for 2 h, filter off the reaction solution, wash it twice with 2400 mL of DMF, add 2400 mL of capping reagent (480 mL of acetic anhydride and 408 mL of pyridine dissolved in 1512 mL of DMF) and react for 2 h, filter off the reaction solution by suction, wash it twice with DMF, DCM, and methanol respectively, and obtain 609.3 g of Fmoc-Gly-Wang after vacuum drying: Take a sample to measure the substitution degree as 0.26 mmol / g.

[0083] Example 2: Synthesis of Fmoc-Ser(tBu)-Wang;

[0084] Place 500.0 g of Wang resin support (sub = 0.42 mmol / g) in a synthesis column, wash it twice with 2400 mL of DMF, add 2400 mL of DCM to swell for 30 min; after filtering off the DCM by suction, add a mixed DCM solution of Fmoc-Ser(tBu)-OH / DIC / HOBT [weigh 312.6 g (400 mmol) of Fmoc-Ser(tBu)-OH and 64.8 g (480 mmol) of HOBT and place them in an amino acid activation bottle, add 2000 mL of a mixed solution of DMF and DCM with a volume ratio of 1:1 and stir to dissolve, add 76.4 ml (480 mmol) of DIC at 0 °C, activate for 5 minutes, add 4.8 g (4 mmol) of DMAP after reacting for 10 min; react for 2 h, filter off the reaction solution, wash it twice with 2400 ml of DMF, add 2400 mL of capping reagent (480 ml of acetic anhydride and 408 ml of pyridine dissolved in 1512 ml of DMF) and react for 2 h, filter off the reaction solution by suction, wash it twice with DMF, DCM, and methanol respectively, and obtain 682.3 g of Fmoc-Ser(tBu)-Wang after vacuum drying: Take a sample to measure the substitution degree as 0.30 mmol / g.

[0085] Example 3: Preparation of the peptide chain fragment 2 resin;

[0086] Accurately weigh 69.23 g (synthesis scale 18 mmol) of Fmoc-Gly-Wang resin with a substitution degree of 0.26 mmol / g in Example 1 and place it in a synthesis column. Add 1000 ml of DCM to swell for 30 min. After filtering off the DCM by suction, wash twice with 800 ml of DMF. Add 1000 ml of 20% piperidine / DMP solution for deprotection twice, reacting for 10 min and 10 min respectively. Then wash twice with 800 ml of DMF, DCM, and DMF respectively. Add 500 ml of a DMF solution containing 25.41 g (36 mmol) of Fmoc-Arg(Pbf)-OH, 5.35 g (39.6 mmol) of HOBT, and 6.3 ml (39.6 mmol) of DIC, stir the reaction with nitrogen bubbling for 2 h. The end point of the reaction is determined by the Kaiser reagent test result. After the reaction reaches the end point, suck out the reaction solution and wash twice with 800 ml of DMF, DCM, and DMF respectively. Subsequently, deprotect again. Repeat the above cyclic operation. According to the sequence of peptide chain fragment 2, couple with protected amino acids one by one. The protected amino acids connected in sequence are: Fmoc-Arg(Pbf)-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. Obtain the peptide chain fragment 2 resin with protected side chains: Fmoc-Gln(Trt)-Ala-Ala-Lys(alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang.

[0087] Example 4: Preparation of peptide chain fragment 4 resin;

[0088] Accurately weigh 75.26 g (synthesis scale 18 mmol) of Fmoc-Ser(tBu)-Wang resin with a substitution degree of 0.30 mmol / g in Example 1 and place it in a synthesis column. Add 1000 ml of DCM to swell for 30 min. After suction filtration to remove DCM, wash with 800 ml of DMF twice. Add 1000 ml of 20% piperidine / DMP solution for deprotection twice, reacting for 10 min and 10 min respectively. Then wash with 800 ml of DMF, DCM, and DMF twice respectively. Add 500 ml of a DMF solution containing 14.61 g (36 mmol) of Fmoc-Thr(Trt)-OH, 5.35 g (39.6 mmol) of HOBT, and 6.3 ml (39.6 mmol) of DIC, stir the reaction under nitrogen bubbling for 2 h. The end point of the reaction is determined by the Kaiser reagent test result. After the reaction reaches the end point, suck out the reaction solution and wash with 800 ml of DMF, DCM, and DMF twice respectively. Subsequently, deprotect again. Repeat the above cyclic operation, and couple with protected amino acids one by one according to the sequence of peptide chain fragment 2. The protected amino acids connected in sequence are: Fmoc-Thr(Trt)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Fmoc-His(Trt)-OH. Obtain peptide chain fragment 4 resin: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Wang.

[0089] Example 5: Preparation of peptide chain fragment 5 resin;

[0090] Accurately weigh 69.23 g (synthesis scale 18 mmol) of Fmoc-Gly-Wang resin with a substitution degree of 0.26 mmol / g in Example 1 and place it in a synthesis column. Add 1000 ml of DCM to swell for 30 min. After filtering off the DCM by suction, wash it twice with 800 ml of DMF. Add 1000 ml of 20% piperidine / DMP solution to deprotect twice, reacting for 10 min and 10 min respectively. Then wash it twice with 800 ml of DMF, DCM, and DMF respectively. Add 500 ml of a DMF solution containing 19.42 g (36 mmol) of Fmoc-Glu(OtBu)-OH, 5.35 g (39.6 mmol) of HOBT, and 6.3 ml (39.6 mmol) of DIC. Stir the reaction under nitrogen for 2 h. The end point of the reaction is determined by the Kaiser reagent test result. After the reaction reaches the end point, suck out the reaction solution and wash it twice with 800 ml of DMF, DCM, and DMF respectively. Then deprotect again. Repeat such cyclic operations and couple with protected amino acids one by one according to the sequence of peptide chain fragment 2. The protected amino acids connected in sequence are: Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH. Obtain peptide chain fragment 5 resin: Fmoc-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Wang.

[0091] Example 6: Method for cleaving the fully protected peptide chain fragment 4 from the resin;

[0092] Put the peptide chain fragment 4 connected to the resin into a mixed solvent of DCM and hexafluoroisopropanol (HFIP). The ratio of the mixed solvent of DCM and hexafluoroisopropanol (HFIP) is 1 - 50:1. The preferred ratio is 5:1. After stirring the mixture at room temperature for 2 - 3 h, filter off the resin. After the filtrate is dried by suction, peptide chain fragment 4 is obtained: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH. After purification, it is used in Example 7.

[0093] Example 7: Connect peptide chain fragment 4 and fragment 5 resin;

[0094] Take the peptide chain fragment 5 resin prepared in Example 5: Fmoc-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Wang, 110 g. Add 1000 ml of 20% piperidine / DMP solution for deprotection twice, reacting for 10 min and 10 min respectively. Then wash it twice with 800 ml of DMF, DCM, and DMF respectively. Add the peptide chain fragment 4 prepared in Example 6: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH, 5.35 g (39.6 mmol) of HOBT, and 500 ml of a DMF solution of 6.3 ml (39.6 mmol) of DIC. Stir the reaction under nitrogen for 2 h. The end point of the reaction is determined by the Kaiser reagent test result. After the reaction reaches the end point, draw off the reaction solution and wash it twice with 800 ml of DMF, DCM, and DMF respectively. Obtain the peptide chain fragment 1 linked to the resin: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Wang.

[0095] Example 8: Method for cleaving the fully protected peptide chain fragment 1 from the resin;

[0096] Put the peptide chain fragment 1 linked to the resin prepared in Example 7 into a mixed solvent of DCM and hexafluoroisopropanol (HFIP). The ratio of the mixed solvent of DCM and hexafluoroisopropanol (HFIP) is 1 - 50:1. The preferred ratio is 5:1. After stirring the mixture at room temperature for 2 - 3 h, filter to remove the resin. After the filtrate is dried by suction, obtain the peptide chain fragment 1: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH. After purification, it is used in Example 9.

[0097] Example 9: Activate the C-terminus of the peptide chain fragment 1;

[0098] Dissolve 50 g of the peptide chain fragment 1 prepared in Example 8 in 200 mL of dry dichloromethane. Add 6.2 g of NHS to the solution, and then add 12.6 g of EDC. Stir and react at 25 °C for 5 hours to form the C-terminal activated fragment 1: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu.

[0099] Example 10: Lys of peptide chain fragment 2 26 Selective deprotection of the protecting group on the side chain;

[0100] Add 1000 ml of DCM to the peptide chain fragment 2 resin prepared in Example 3 and wash it twice, then add 1000 ml of DCM, slowly add 27 ml of phenylsilane while stirring the resin evenly. After reacting for 3 min, add 5.64 g of Pd(PPh3)4 and react at room temperature for 50 min. Drain the reaction solution, and treat the above resin with a hydrazine hydrate / DMF (1 / 15) mixed solution 3 times. Wash it 6 times with 1000 ml of DCM: Take a sample for Kaiser reagent detection, and the resin shows blue. Thus, Lys is obtained. 26 Peptide chain fragment 2 with side chain deprotected and attached to the resin: Fmoc-Gln(Trt)-Ala-Ala-Lys 26 -Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang.

[0101] Example 11: Lys 26 Method for cleaving the deprotected peptide chain fragment 2 from the resin;

[0102] Put the Lys 26 Deprotected peptide chain fragment 2 prepared in Example 10 into a mixed solvent of DCM and hexafluoroisopropanol (HFIP). The ratio of the mixed solvent of DCM and hexafluoroisopropanol (HFIP) is 1 - 50:1. The preferred ratio is 5:1. After stirring the mixture at room temperature for 2 - 3 hours, filter to remove the resin. After draining the filtrate, the peptide chain fragment: Fmoc-Gln(Trt)-Ala-Ala-Lys 26 -Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH is obtained. Without purification, it can be used in Example 12.

[0103] Example 12: Lys in liquid phase 26 Lys of the deprotected peptide chain fragment 2 26 Fatty acylation of the side chain;

[0104] Weigh 28.9 g of tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu and 2.67 g (19.8 mol) of HOBT into a dissolution flask, add 500 ml of DMF to dissolve. After complete dissolution, place it in an ice-water bath and let it stand for 10 min. Then add 3.1 ml (19.8 mmol) of DIC, mix well, and activate it in the ice-water bath for 10 min. Add the activated solution to 200 mL of the DMF solution of Fmoc-Gln(Trt)-Ala-Ala-Lys 26 -Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH prepared in Example 11, and stir the reaction evenly. Monitor the reaction by TLC. After the reaction is completed, drain the solvent, and purify it by preparative HPLC to obtain Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH, weighing 76.3 g.

[0105] Example 13: Preparation of fragment 3;

[0106] Take 50 g of the peptide chain Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH prepared in Example 12 and dissolve it in 200 mL of DMF solution. Then add 500 ml of 20% piperidine / DMF solution for deprotection, and react for 20 min. After the reaction is completed, drain the solvent, and purify it by preparative HPLC. Thus, fragment 3: Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH is obtained. It is used in Example 13.

[0107] Example 14: Assembly of the semaglutide peptide chain;

[0108] Weigh the peptide chain fragment Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic Acid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH prepared in Example 13 and the C-terminally activated peptide chain Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Osu prepared in Example 9, add them to 500 ml of DMF, and stir under nitrogen bubbling at room temperature for 2 hours. Remove the solvent under reduced pressure, and after purification, obtain the peptide chain fragment Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic Acid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

[0109] Example 14: Deprotection of semaglutide;

[0110] Add the semaglutide peptide chain obtained in Example 13 to 1600 ml of frozen cleavage solution (volume ratio of TFA / thioanisole / TIS / 120 = 92.5 / 2.5 / 2.5 / 2.5), and stir and react at room temperature for 4 h. After the cleavage reaction is completed, filter the resin, wash the resin twice with 100 ml of TFA, combine the filtrate and the washing solution, concentrate by rotary evaporation to 1000 ml, pour it into 10 L of frozen methyl tert-butyl ether, and precipitate a white solid; after standing for 30 min, filter, wash with methyl tert-butyl ether 6 times, and dry under vacuum to obtain 72.3 g of crude semaglutide, with a crude peptide yield of 96.8% and a purity of 93.8%.

[0111] Example 15: Purification of semaglutide;

[0112] 50.0 g of the crude peptide prepared in Example 12 was dissolved in 200 ml of acetic acid. After complete dissolution, it was diluted with water to 4000 ml. The solution was filtered through a 0.45 μm filter membrane for standby. Purification method: a C18 preparative column with a diameter of 150 mm, the mobile phase was a 0.1% acetic acid / water - 0.1% acetic acid / acetonitrile system, the sample loading amount was 25 g / time, the flow rate was 300 ml / min, and gradient elution was performed; pre-peak and post-peak cyclic injection was carried out to obtain a refined peptide solution with qualified central control analysis purity. After desalting, it was freeze-dried to obtain 29.5 g of refined peptide, with a purity of over 99.6% and a single impurity of less than 0.1%.

[0113] The crude peptide of semaglutide prepared by this synthesis method has both high yield and high purity. Through conventional purification means, semaglutide refined peptide with a purity of over 99.6% and a single impurity of less than 0.1% can be obtained with a high yield.

[0114] In the present invention, the main chain is divided into 3 shorter polypeptide fragments at positions 7 - 14, 15 - 22, and 23 - 37 and synthesized simultaneously. The 7 - 14 peptide segment (fragment 4) and the 15 - 22 peptide segment (fragment 5) are assembled into a 7 - 22 peptide segment (fragment 1). On the other hand, after the 23 - 37 peptide segment (fragment 2) is assembled, the protecting group A11oc on Lys 26 is selectively removed. In the liquid phase system, the side chain of Lys 26 is fatty acidified to form fragment 3. Then, the 7 - 22 peptide segment and the 23 - 37 peptide segment are connected in the liquid phase reaction system. After separation and purification, the protecting groups are removed to obtain the crude product. Then, through separation and purification, the finished product is obtained. This method can not only make full use of the characteristics of high efficiency and easy purification of solid-phase synthesis, but also break up the fragment with a large number of hydrophobic amino acids between His 7 and Lys 26 into smaller parts to avoid the formation of serious β-sheets and resin polycondensation. On the other hand, the liquid phase reaction system is used for the coupling of the 7 - 22 peptide segment and the 23 - 37 peptide segment, and the fatty acidification of the side chain of Lys 26 in peptide chain fragment 2. In the liquid phase system, both reaction substrates participate in the reaction in a molar ratio close to 1:1. Compared with the traditional solid-phase synthesis method (usually requiring 1:3 - 8), it has significant economic advantages.

[0115] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0116] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A semaglutide, characterized in that: The following method is used for synthesis, and the specific steps are as follows: 1) Splitting the semaglutide backbone into three polypeptide fragments: positions 7-14, 15-22, and 23-37; 2) preparing the 7-14 fragment, the 15-22 fragment and the 23-37 fragment respectively by solid phase synthesis; 3) connecting the 7-14 fragment and the 15-22 fragment to form a 7-22 fragment, and activating the C-terminus of the fragment to an active ester; 4) Selectively remove Lys in the 23-37 fragment 26 The Alloc protecting group is then added and then fatty acid modified to obtain a modified 23-37 fragment; 5) in a liquid phase system, coupling the activated 7-22 fragment and the modified 23-37 fragment at a molar ratio of 1:0.6-1.4 to form a complete polypeptide chain; 6) removing the protecting group and purifying to obtain the finished product of semaglutide.

2. A semaglutide according to claim 1, characterized in that: The division of the polypeptide fragments in step (1) is specifically as follows: The 7-14 fragment sequence contains: His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-(Trt)-Ser(tBu)-Asp(OtBu); The sequence of the fragment 15-22 contains: Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly; The fragment sequence from position 23 to 37 contains: Gln(Trt)-Ala-Ala-Lys(Alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly.

3. The semaglutide according to claim 1, characterized in that: The solid phase synthesis in step (2) uses Wang resin with a substitution degree of 0.1-0.6 mmol / g as a carrier, and the condensing agent in the coupling reaction is selected from at least one of DIC / HOBT, DIC / HOAT, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, and HATU / HOAT / DIPEA.

4. The semaglutide according to claim 1, characterized in that: The conditions for selectively removing the Alloc protecting group in step (4) are: using 0.05-0.5 equivalents of Pd(PPh3)4 and 5-50 equivalents of phenylsilane or morpholine, and the reaction time is 10-120 minutes.

5. The semaglutide according to claim 1, characterized in that: The C-terminal activation in step (3) is achieved by the following method: The 7-22 fragment is dissolved in dichloromethane or DMF, and N-hydroxysuccinimide (NHS) and coupling reagent DCC or EDC are added, and the reaction is carried out at 10-60° C. for 0.5-24 hours to form NHS ester.

6. A semaglutide according to any one of claims 1 to 5, characterized in that: After the coupling reaction in step (5) is completed, the reagent for cleaving the protecting group is a TFA solution containing 1-10% of a scavenger, wherein the scavenger is selected from at least two of anisole, thioanisole, ethanedithiol, mercaptoethanol, phenol, water, and triisopropylsilane (TIS).

7. The semaglutide according to claim 1, characterized in that: TFA 85-95%, thioanisole 1-5%, water 1-10%, TIS 1-5%.

8. The semaglutide according to claim 1, characterized in that: The deprotection reagent for solid phase synthesis in step (2) is a piperidine / DMF solution with a volume ratio of 15-25%, and the deprotection time is 1-30 minutes.

9. The semaglutide according to claim 1, characterized in that: The connection between the 7-14 fragment and the 15-22 fragment in step (3) is achieved by reacting the 15-22 fragment resin removed from the Fmoc protection with the 7-14 fragment of the free carboxylic acid in the presence of a condensing agent, wherein the amount of the condensing agent is 2-10 times the molar amount of the fragment.

10. The semaglutide according to claim 1, characterized in that: The purification in step (6) adopts high performance liquid chromatography, the mobile phase is an acetonitrile / water mixed solution, the volume proportion of acetonitrile is 10-90%, and the purification temperature is 10-40°C.

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