A method for synthesizing high purity retatrutide

Abstract

This invention belongs to the field of peptide drug synthesis, specifically relating to a method for synthesizing high-purity retatrutide. This invention utilizes fragments such as Fmoc-Ile-Glu(OtBu)-OH for synthesis, effectively avoiding the generation of Ile-deficient impurities and reducing the use of expensive materials. By optimizing the synthesis process, this invention reduces the synthesis difficulty and shortens the product production cycle. The resulting crude retatrutide has high purity, facilitating subsequent preparation and purification, and significantly improving product purity and yield.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drug synthesis, specifically relating to a method for synthesizing high-purity Retatrutide. Background Technology

[0002] Retatrutide, developed by Eli Lilly, is a triple receptor agonist targeting GLP-1 (glucagon-like peptide-1), GIP (glucose-dependent insulinotropic peptide), and GCG (glucagon). Clinical trials have shown that Retatrutide is effective in treating non-alcoholic fatty liver disease, overweight, type 2 diabetes, and reducing the incidence of cardiovascular events. Retatrutide is the first GLP-1 / GIP / GCG triple receptor agonist drug to enter Phase III clinical trials. Most Wall Street analysts agree that the market for GLP-1-based weight-loss drugs will be enormous, with multiple institutions predicting that annual sales could reach approximately $100 billion by 2030.

[0003] Retatrutide is a long-acting polypeptide consisting of 39 amino acids modified with fatty acid acylation. It is designed with a GIP peptide backbone and has an average half-life of up to 6 days in humans and 21 hours in mice. Its sequence structure is: H-Tyr 1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 - Ser 11 -Ile 12 -αMeLeu 13 -Leu 14 -Asp 15 -Lys 16 -Lys 17 (AEEA-γ-Glu-Eicosanedioic acid)-Ala 18 -Gln 19 -Aib 20 -Ala 21 -Phe 22 -Ile 23 -Glu 24 -Tyr 25 -Leu 26 -Leu 27 -Glu 28 -Gly29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -NH2. The compound is disclosed in patent application WO2019125938A1.

[0004] Currently, research on the synthetic process of this polypeptide is still relatively limited. Due to its long sequence and complex structure, existing synthetic methods generally suffer from problems such as long production cycles and low purity of intermediates and crude products. In stepwise synthesis, racemic amino acid impurities are easily generated due to long reaction times, and sequence deletions (especially Ile) are also common. 23 The presence of byproducts such as peptide fragments (such as their location) makes purification difficult and results in low yields. Furthermore, the high cost of using peptide fragment raw materials hinders large-scale production.

[0005] Therefore, there is an urgent need in the field to develop a synthetic method for Retatrutide that has a short synthesis cycle, is easy to operate, can effectively control racemic and deletion impurities, improve the purity of crude product, and ultimately achieve high-purity refined peptides with high purification yield, so as to meet the needs of its clinical and commercial production. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a method for synthesizing high-purity Retatrutide.

[0007] This invention provides a method for synthesizing retatrutide, comprising: Peptide resins were prepared by sequential coupling according to the Retatrutide sequence; The peptide resin was cleaved to obtain Retatrutide; Among them, Ile 23 -Glu 24 The coupling raw material is Fmoc-Ile-Glu(OtBu)-OH.

[0008] In the prior art, Ile is directly coupled sequentially. 23 and Glu 24 At that time, due to issues such as spatial steric hindrance or coupling efficiency, missing Ile is easily generated. 23 Impurities. Through experimental screening and optimization, compared to directly coupling Ile sequentially... 23 and Glu 24This invention uses the Fmoc-Ile-Glu(OtBu)-OH dipeptide fragment to... 23 and Glu 24 Pre-condensation before incorporation into the main chain can effectively avoid the generation of this missing peptide impurity, while reducing the use of expensive materials and improving product purity and yield.

[0009] In some embodiments, the sequential coupling includes segmented coupling, and the coupling order is: Ser, Pro-Pro, Pro, Ala, Gly, Ser, Ser, Pro, Gly-Gly, Glu, Leu, Leu, Tyr, Ile-Glu, Phe, Ala, Aib, Gln, Ala, Lys[C20-γGlu-AEEA], Lys, Asp, Leu, α-Me-Leu, Ile, Ser, Tyr, Asp, Ser, Thr, Phe, Thr, Gly, Gln, and Tyr-Aib.

[0010] Through experimental screening, it was determined that, compared with the traditional method of coupling individual amino acids one by one, the present invention adopts the above-mentioned segmented coupling strategy, which introduces multiple dipeptide fragments (such as Pro-Pro, Gly-Gly, Ile-Glu, Tyr-Aib, etc.), which can significantly reduce coupling steps, shorten the synthesis cycle, reduce the risk of racemic side reactions caused by long chain elongation, and improve the purity of crude product.

[0011] In some embodiments, the Tyr 1 -Aib 2 The coupling raw materials are Boc-Tyr(tBu)-Aib-OH, Gln 3 The coupling raw materials are Fmoc-Gln(Trt)-OH and Gly 4 The coupling raw material is Fmoc-Gly-OH,Thr 5 The coupling raw materials are Fmoc-Thr(tBu)-OH and Phe 6 The coupling raw material is Fmoc-Phe-OH,Thr 7 The coupling raw materials are Fmoc-Thr(tBu)-OH and Ser 8 The coupling raw materials are Fmoc-Ser(tBu)-OH and Asp. 9 The coupling raw material is Fmoc-Asp(OtBu)-OH, Tyr 10 The coupling raw materials are Fmoc-Tyr(tBu)-OH and Ser 11 The coupling raw material is Fmoc-Ser(tBu)-OH, Ile 12 The coupling raw materials are Fmoc-Ile-OH and αMeLeu. 13The coupling raw material is Fmoc-[α-Me-Leu]-OH, Leu 14 The coupling raw materials are Fmoc-Leu-OH and Asp. 15 The coupling raw materials are Fmoc-Asp(OtBu)-OH and Lys 16 The coupling raw material is Fmoc-Lys(Boc)-OH, Lys 17 The coupling agent for [C20-γGlu-AEEA] is Fmoc-Lys[C20-OtBu-γGlu(OtBu)-AEEA]-OH, Ala 18 The coupling raw materials are Fmoc-Ala-OH and Gln. 19 The coupling raw materials are Fmoc-Gln(Trt)-OH and Aib. 20 The coupling raw materials are Fmoc-Aib-OH and Ala 21 The coupling raw materials are Fmoc-Ala-OH and Phe 22 The coupling raw materials are Fmoc-Phe-OH and Ile. 23 -Glu 24 The coupling raw material is Fmoc-Ile-Glu(OtBu)-OH, Tyr 25 The coupling raw materials are Fmoc-Tyr(tBu)-OH and Leu 26 The coupling raw material is Fmoc-Leu-OH, Leu 27 The coupling raw materials are Fmoc-Leu-OH and Glu. 28 The coupling raw materials are Fmoc-Glu(OtBu)-OH and Gly 29 -Gly 30 The coupling raw material is Fmoc-Gly-Gly-OH, Pro 31 The coupling raw materials are Fmoc-Pro-OH and Ser 32 The coupling raw material is Fmoc-Ser(tBu)-OH, Ser 33 The coupling raw materials are Fmoc-Ser(tBu)-OH and Gly 34 The coupling raw materials are Fmoc-Gly-OH and Ala 35 The coupling raw material is Fmoc-Ala-OH, Pro 36 The coupling raw material is Fmoc-Pro-OH, Pro 37 -Pro 38 The coupling raw materials are Fmoc-Pro-Pro-OH and Ser 39 The coupling raw material is Fmoc-Ser(tBu)-OH.

[0012] Through experimental optimization, it was determined that, compared to other protected amino acids or fragments, using the aforementioned amino acids or fragments with specific side-chain protecting groups (such as tBu, OtBu, Trt, or Boc) can ensure the stability of the side chains during solid-phase synthesis and their effective and synchronous removal in the final cleavage step. Among these, Boc-Tyr(tBu)-Aib-OH, as an N-terminal dipeptide fragment, can avoid the adverse effects on the N-terminus during conventional Fmoc deprotection, thus improving the yield and purity of the final product.

[0013] In some embodiments, the resin is an amino resin selected from Rink Amide AM Resin, Rink Amide MBHA Resin, or Sieber Resin. Specifically, the resin is Rink Amide-AM Resin.

[0014] In some embodiments, the resin has a crosslinking degree of 1% to 2%, a particle size of 100 to 200 mesh, and a substitution degree of 0.3 to 0.8 mmol / g.

[0015] Through experimental screening, compared with other types of resins (such as Sieber Resin) or resins with different parameters, the Rink Amide AMResin selected in this invention with a degree of substitution of 0.3~0.8 mmol / g, a degree of crosslinking of 1%~2%, and a particle size of 100~200 mesh can provide suitable loading and good swelling properties, which is conducive to the growth of long peptide chains on the resin, reduces coupling difficulties caused by steric hindrance, and ensures that the peptide chains can be completely cleaved from the resin during final cleavage.

[0016] In some specific embodiments, the resin has a crosslinking degree of 1%, a particle size of 100-200 mesh, and a substitution degree of 0.5 mmol / g.

[0017] In some embodiments, the amino acid equivalent of the coupling raw material is 2.0 to 3.0 eq. For example, the amino acid equivalent of the coupling raw material is 2.0, 2.3, 2.5, 2.8, or 3.0 eq. Specifically, the amino acid equivalent of the coupling raw material is 3.0 eq.

[0018] Through experimental screening, it was determined that, compared with the problems of incomplete coupling when the amount of coupling raw material is less than 2.0 times and serious material waste when it is more than 3.0 times, the amount used in this invention ensures coupling efficiency (ninhydrin test is negative) while taking into account cost control, making it suitable for industrial scale-up production.

[0019] In some embodiments, the coupling includes the steps of removing the N-terminal protecting group and condensation.

[0020] In some embodiments, the reagent for removing the N-terminal protecting group includes a mixed solution of piperidine and DMF, wherein the volume percentage of piperidine is 20%, the volume percentage of DMF is 80%, and the reaction time for removing the N-terminal protecting group is 15 min.

[0021] Through experimental optimization, it was determined that, compared with other deprotection times (such as <10 min which is prone to incomplete removal, and >20 min which increases the risk of side reactions), the present invention uses 20% piperidine / DMF treatment for 15 min, which can efficiently and completely remove the Fmoc protecting group. The result was positive by ninhydrin detection, providing sufficient reaction sites for subsequent coupling.

[0022] In some embodiments, the condensation reagent is selected from one or more of DIC / HOBt, DIC / Oxyma Pure, HBTU / DIEA, or PyBop / DIEA, and the activation time for condensation is 5 min. Specifically, the condensation reagent is DIC / Oxyma Pure.

[0023] Experimental results show that, compared to traditional single condensation systems or schemes with excessively long activation times, the condensation reagent used in this invention can complete rapid activation within 5 minutes, effectively reducing the occurrence of racemic side reactions, improving coupling efficiency, and ensuring the accuracy of peptide chain elongation.

[0024] In some embodiments, the coupling reaction solvent is selected from one or more of DCM, DMF, NMP, or DMSO, the reaction time is 2-3 h, and the reaction temperature is 20-30 °C. For example, the reaction time is 2, 2.5, or 3 h, and the reaction temperature is 20, 22, 24, 26, 28, or 30 °C.

[0025] Through experimental screening and optimization, this invention employs a reaction time of 2-3 hours, which, compared to methods where excessively short reaction times lead to incomplete coupling or excessively long reaction times increase the risk of racemization, ensures complete coupling while maintaining synthetic efficiency. Conversely, compared to methods where excessively low reaction temperatures result in decreased coupling rates and incomplete reactions, or excessively high reaction temperatures increase the risk of racemic side reactions and removal of side-chain protecting groups, this invention uses a reaction temperature of 20-30°C, which effectively suppresses the formation of racemic products while maintaining coupling efficiency and improving the purity of the target peptide chain. These reaction conditions are compatible with the selected solvent system (DMF, etc.), maintaining good solubility of the amino acid derivatives while avoiding resin degradation or side reactions that may occur at high temperatures, thus contributing to process stability and consistency in scale-up production.

[0026] In some embodiments, the reagents used in the peptide resin cleavage include trifluoroacetic acid, 1,2-ethylenedithiol, triisopropylsilane, and water; wherein the volume ratio of trifluoroacetic acid, 1,2-ethylenedithiol, triisopropylsilane, and water is (80~100):(1~10):(0.5~5):(0.5~5), the reaction time is 2~3 h, and the reaction temperature is 20~30 °C.

[0027] For example, the volume ratio of trifluoroacetic acid, 1,2-ethylenedithiol, triisopropylsilane, and water is 80:1:0.5:0.5, 85:1.5:1:1.5, 90:5:2.5:2.5, 95:7:3:4, or 100:10:5:5. Specifically, the volume ratio of trifluoroacetic acid, 1,2-ethylenedithiol, triisopropylsilane, and water is 90:5:2.5:2.5.

[0028] Through experimental screening and optimization, this invention uses the same reagent for both peptide resin cleavage and side-chain protecting group removal, allowing cleavage and deprotection to occur simultaneously. Compared to using different reagents, this simplifies the operation process, shortens reaction time, and improves synthesis efficiency. Furthermore, compared to a single TFA cleavage system, the reagent used in this invention, under a volume ratio of 90:5:2.5:2.5 (as in Example 1), can simultaneously and efficiently break resin linkages and remove side-chain protecting groups, achieving a crude peptide purity of over 77.34% and a total yield of 72.43%, significantly reducing side reactions.

[0029] In some embodiments, the synthesis method further includes a precipitation step.

[0030] In some embodiments, the precipitating agent includes anhydrous methyl tert-butyl ether. Through experimental screening, it was determined that using anhydrous methyl tert-butyl ether as a precipitating agent, compared to other precipitating agents, can more effectively remove organic impurities from the lysis buffer, yielding a loose, easily dried crude peptide solid, which is beneficial for subsequent purification operations.

[0031] In some embodiments, the synthesis method further includes a purification step, wherein the purification conditions include: The chromatographic column is a C18 column; Mobile phase A is an acidic aqueous solution, and mobile phase B is an acetonitrile solution or an acidic acetonitrile solution; The acidic aqueous solution is selected from a trifluoroacetic acid aqueous solution with a volume percentage of 0.03% to 2.0%, or an acetic acid aqueous solution with a volume percentage of 0.3% to 5.0%. The acidic acetonitrile solution includes an acetic acid-acetonitrile solution, wherein the volume percentage of acetic acid is 0.5-1.5%.

[0032] Experiments have shown that, compared to other reagents, using a lower concentration of trifluoroacetic acid aqueous solution can effectively separate hydrophobic impurities during purification, while using acetic acid aqueous solution is beneficial for the product to be converted into a more stable acetate form during purification.

[0033] In some embodiments, the purification includes a first purification and a second purification. The first purification mainly removes impurities with large polarity differences, while the second purification further refines and completes the salt form conversion.

[0034] In some embodiments, the mobile phase A for the first purification is an aqueous solution of trifluoroacetic acid with a volume percentage of 0.03% to 2.0%, and the mobile phase B is an acetonitrile solution. For example, the volume percentage of the aqueous trifluoroacetic acid solution is 0.03%, 0.05%, 0.1%, 0.5%, 1.0%, 1.5%, or 2.0%. Specifically, the volume percentage of the aqueous trifluoroacetic acid solution is 0.1%.

[0035] In some embodiments, the mobile phase A of the second purification is an aqueous acetic acid solution with a volume percentage of 0.3% to 5.0%, and the mobile phase B is an acetic acid-acetonitrile solution, wherein the volume percentage of acetic acid is 0.5% to 1.5%; for example, the volume percentage of the aqueous acetic acid solution is 0.3%, 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, or 5.0%; and the volume percentage of acetic acid in the acetic acid-acetonitrile solution is 0.5%, 0.8%, 1.0%, 1.2%, or 1.5%. Specifically, the volume percentage of the aqueous acetic acid solution is 3%; and the volume percentage of acetic acid in the acetic acid-acetonitrile solution is 1%.

[0036] Through experimental screening and optimization, compared with a single mobile phase system, this invention adopts a two-step purification process of primary purification (0.1% TFA / water-acetonitrile) and secondary purification (3% acetic acid aqueous solution-1% acetic acid-acetonitrile solution), which can remove strongly hydrophobic impurities and ion pair residues successively, thereby increasing the purity of the final product to over 99.2% and controlling single impurities to below 0.1%.

[0037] In some embodiments, the elution method is gradient elution, and the elution procedure is specifically as follows: From 0 to 10 minutes, the volume fraction of phase A was 90%, and the volume fraction of phase B was 10%. Over 10-15 minutes, the volume fraction of phase A decreased from 90% to 60%, and the volume fraction of phase B decreased from 10% to 40%. Over 15 to 80 minutes, the volume fraction of phase A decreased from 60% to 45%, and the volume fraction of phase B decreased from 40% to 55%.

[0038] Experiments have verified that, compared to isocratic elution or other gradient ranges, the elution procedure described above in this invention can effectively separate the Retatrutide main peak from the adjacent impurity peaks. This gradient range is the optimal elution procedure optimized for the specific physicochemical properties of Retatrutide, resulting in high recovery rate, high product purity, and single impurity <0.1%.

[0039] The present invention also provides Retatrutide, which is prepared by the synthesis method described above.

[0040] Furthermore, the present invention provides the use of Retatrutide as described above in the preparation of medicaments for treating type 2 diabetes, obesity, or non-alcoholic steatohepatitis.

[0041] Furthermore, the present invention also provides a method for treating type 2 diabetes, obesity, or non-alcoholic steatohepatitis, comprising administering a therapeutically effective amount of Retatrutide as described above to a subject in need.

[0042] This invention relates to a method for synthesizing high-purity retatrutide. It addresses the issue that existing retatrutide synthesis processes are prone to Ile deletion. 23 Addressing issues such as site-specific impurities, long synthesis cycles, and low purification yields, this invention optimizes the synthesis process by employing the Fmoc-Ile-Glu(OtBu)-OH dipeptide fragment for coupling, effectively avoiding the generation of Ile-deficient impurities and reducing the use of expensive materials. The method provided by this invention reduces synthesis difficulty, shortens product production cycles, and offers stable processes conducive to industrial scale-up. The resulting crude Retatrutide product exhibits high purity, facilitating subsequent purification and significantly improving product purity and overall yield. Detailed Implementation

[0043] This invention provides a method for synthesizing high-purity retatrutide. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0044] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0045] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0046] When used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0047] The term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0048] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0049] A method for preparing retatrutide, comprising: Preparation of peptide resin: Protected amino acids or protected polypeptide fragments are coupled one by one onto a solid-phase support resin according to the sequence of Retatrutide to obtain peptide resin.

[0050] Preparation of crude peptides: The peptide resin was lysed using a lysis buffer to obtain crude peptides.

[0051] Preparation of refined product: The crude peptide is purified to obtain Retatrutide.

[0052] Among them, Ile 23 -Glu 24 The Fmoc-Ile-Glu(OtBu)-OH fragment was used.

[0053] In this step, the protected polypeptide fragment is selected from at least one of Fmoc-Pro-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ile-Glu(OtBu)-OH, Fmoc-Lys[C20-OtBu-γGlu(OtBu)-AEEA]-OH, and Boc-Tyr(tBu)-Aib-OH.

[0054] The detailed technical solution of the method for synthesizing high-purity retatrutide according to the present invention is as follows: Amino acid resin was selected, and after swelling with solvent, the solvent was dried, Fmoc was removed with deprotection reagent, the solvent was dried again, and the resin was washed. The resin tested positive for ninhydrin. After reacting with amino acids activated with condensing agent, the resin tested negative for ninhydrin, and then dried and washed.

[0055] According to the amino acid sequence Fmoc-Ser(tBu)-OH, Fmoc-Pro-Pro-OH, Fmoc-Pro-OH , Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-T yr(tBu)-OH, Fmoc-Ile-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Aib-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Lys[C20-OtBu-γ-Glu(OtBu)-AEEA]-OH The following peptides were coupled together to obtain a fully protected peptide resin: Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-[α-Me-Leu]-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, and Boc-Tyr(tBu)-Aib-OH.

[0056] The crude Retatrutide peptide was obtained by adding a lysis reagent to the fully protected peptide resin, followed by precipitation and drying.

[0057] The crude peptide was purified to obtain Retatrutide.

[0058] The amino resin selected is Rink Amide AM Resin, Rink Amide MBHA Resin or Sieber Resin, with a crosslinking degree of 1~2%, a particle size of 100~200 mesh, and a resin substitution degree of 0.3~0.8mmol / g.

[0059] Pro 37 -Pro 38Using Fmoc-Pro-Pro-OH; Gly 29 -Gly 30 Using Fmoc-Gly-Gly-OH; Ile 23 -Glu 24 Using Fmoc-Ile-Glu(OtBu)-OH; Lys 17 Using Fmoc-Lys[C20-OtBu-γGlu(OtBu)-AEEA]-OH;Tyr 1 -Aib 2 Boc-Tyr(tBu)-Aib-OH was used.

[0060] The amino acid equivalent of the Fmoc protected amino acid or the protected amino acid fragment is 2.0~3.0 eq.

[0061] The reaction solvent is one or more of DCM, DMF, NMP, and DMSO; The condensing agent includes one or more of DIC / HOBt, DIC / Oxyma Pure, HBTU / DIEA, and PyBop / IDIEA.

[0062] The Fmoc removal reagent is a mixed solution of piperidine and DMF; The Fmoc removal reagent consists of 20% piperidine and 80% DMF by volume.

[0063] The reagents used for peptide resin cleavage include TFA, EDT, TIS and water, with a volume ratio of (80~100):(1~10):(0.5~5):(0.5~5). During this cleavage reaction, the breaking of peptide resin linkages and the removal of side chain protecting groups are completed simultaneously.

[0064] The purification method includes: using C18 silica gel packing as the stationary phase, acidic aqueous solution as phase A, and acetonitrile as phase B, performing gradient elution; The purification method further includes: using octadecylsilane-bonded silica gel as the stationary phase, and employing a 0.03%–2.0% TFA aqueous solution as phase A and an acetonitrile solution as phase B for the first purification, collecting the fraction with a purity greater than 95%. Then, a second purification is performed using a 0.3%–5.0% HOAc aqueous solution as phase A and an acetic acid-acetonitrile solution as phase B, collecting the fraction with a purity greater than 99%. Finally, the product is obtained by lyophilization after column salting or direct solution salting.

[0065] This method has a high product yield and low cost, making it suitable for industrial-scale production.

[0066] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0068] The present invention will be further illustrated below with reference to the embodiments: Example 1 (1) Preparation of peptide resin 9.8 g of Rink Amide-AM Resin resin (Sub=0.51 mmol / g) was weighed and added to a solid-phase reactor, followed by 80 mL of DCM swelling grease for 0.5 h. The solvent was then removed by vacuum, and 80 mL of a v / v 20% piperidine / DMF solution (i.e., a DMF solution containing 20% ​​piperidine by volume) was added. The deprotection reaction was allowed to proceed for 15 min. The solution was then removed by vacuum, and 80 mL of DMF was added for washing. The ninhydrin test result was positive.

[0069] Weigh 5.75 g of Fmoc-Ser(tBu)-OH, 2.13 g of Oxyma, 1.89 g of DIC, and 60 mL of DMF solution. Activate in an ice bath for 5 minutes, with the activation temperature not exceeding 10°C. Add the activated solution to the reactor and react for 2 hours. After a negative result for ninhydrin, dry the solution under vacuum. Wash three times with 80 mL of DMF each time.

[0070] Repeat the above steps and follow the amino acid sequence Fmoc-Pro-Pro-OH, Fmoc-Pro-OH , Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmo c-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Aib-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Lys[C20-OtBu-γGlu( The following peptides were coupled sequentially: [OtBu]-AEEA-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-[a-Me-Leu]-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, and Boc-Tyr(tBu)-Aib-OH. After coupling, the peptide resin was washed six times with 100 mL of methanol each time. It was then dried under vacuum to obtain the peptide resin. Among them, Fmoc-Ile-Glu(OtBu)-OH(Ile) 23 -Glu 24 The amino acid equivalent of the mixture is 3 eq, the coupling reaction temperature is 20~30℃, and the reaction time is 2~3h.

[0071] (2) Preparation of crude product

[0072] 400 mL of lysis reagent (TFA:EDT:TIS:water volume ratio = 90:5:2.5:2.5) was prepared. The fully protected peptide resin was added under ice bath conditions, and the reaction was continued for 2 hours at room temperature. During this lysis reaction, the breaking of the peptide resin linkage bonds and the removal of the side-chain protecting groups occurred simultaneously. After the reaction was complete, anhydrous methyl tert-butyl ether was added to precipitate the peptide. After centrifugation and drying, the product obtained was the crude Retatrutide peptide, yielding 23.85 g. The crude peptide was quantified to 17.13 g using a reference standard, resulting in a total yield of 72.43% and a purity greater than 77.34%.

[0073] (3) Preparation of high-quality products

[0074] Take 23.8g of crude peptide and dissolve it in 2000mL of solvent (20% acetonitrile aqueous solution). Purify it using a 200mm × 250mm column with C18 packing material. For the first purification, the mobile phase is: Phase A: 0.1% TFA / water solution; Phase B: acetonitrile, with an elution gradient of 40% Phase B to 55% Phase B. The specific elution program is shown in Table 1.

[0075] Table 1 Elution Procedure

[0076] The collected liquid was tested and the fractions with a purity greater than 98% were combined for a second purification. The second purification phase A consisted of 3% acetic acid aqueous solution, and the second phase B consisted of 1% acetic acid acetonitrile solution. The elution gradient was 40% phase B to 55% phase B. The specific elution procedure is shown in Table 2.

[0077] Table 2 Elution Procedure

[0078] The salt was replaced by a phase-changing flow, concentrated, and then freeze-dried to obtain 9.53 g of refined Retatrutide with a purity greater than 99.2%, a single impurity of less than 0.1%, and an overall yield of 40%.

[0079] Comparative Example

[0080] Weigh 9.8 g of Rink Amide-AM Resin resin (Sub=0.51 mmol / g) and add it to a solid-phase reactor. Add 80 mL of DCM swelling grease and incubate for 0.5 h. Remove the solvent and add 80 mL of a 20% piperidine / DMF solution (v / v, DMF solution containing 20% ​​piperidine by volume). Perform a deprotection reaction for 15 min. Remove the solvent and wash with 80 mL of DMF. The ninhydrin test result is positive. Weigh 5.75 g of Fmoc-Ser(tBu)-OH, 2.13 g of Oxyma, 1.89 g of DIC, and 60 mL of DMF solution. Activate in an ice bath for 5 min, with the activation temperature not exceeding 10°C. Add the activated solution to the reactor and react for 2 h. Perform a ninhydrin test. After a negative result, remove the solvent and wash with 80 mL of DMF three times.

[0081] Repeat the above steps and follow the amino acid sequence Fmoc-Pro-Pro-OH, Fmoc-Pro-OH , Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Tyr(t Bu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Aib-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Lys[C20-OtBu-γGlu(OtBu)-AEEA]-OH , Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-[a-Me-Leu]-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Boc-Tyr(tBu)-Aib-OH.

[0082] In the comparative examples, different condensation methods were used for the 23rd amino acid Fmoc-Ile-OH, as described in the comparative examples below.

[0083] Comparative Example 1: The amino acid equivalents of Fmoc-Ile-OH (position 23) were determined using 6 eq (10.60 g), HATU 11.41 g, DIEA 4.26 g, and 60 mL of LDMF solution, activated in an ice bath for 5 minutes. The reaction was carried out at 30 °C for 4 hours to complete sequence coupling, yielding peptide resin. The lysis reagent was TFA:EDT:TIS:water (volume ratio) of 90:5:2.5:2.5. After lysis, MTBE precipitated, crystallized, and analyzed by solid-state HPLC. The purity was 58.12%, with Ile missing. 23 Impurities: 18.39%.

[0084] Comparative Example 2: The amino acid equivalents of Fmoc-Ile-OH (position 23) were determined using 6 eq (10.60 g), PyBop 15.61 g, DIEA 4.26 g, and 60 mL of LDMF solution, activated in an ice bath for 5 minutes. The reaction was carried out at 50 °C for 4 hours to complete sequence coupling, yielding peptide resin. The lysis reagent was TFA:EDT:TIS:water (volume ratio) of 90:5:2.5:2.5. After lysis, MTBE precipitated, crystallized, and analyzed by solid-state HPLC. The purity was 55.43%, with Ile missing. 23 Impurities: 17.21%.

[0085] Comparative Example 3: The amino acid equivalents of Fmoc-Ile-OH (position 23) were determined using 3 eq (5.31 g), Oxyma 2.13 g, DIC 1.89 g, and 60 mL of LDMF solution, activated in an ice bath for 5 minutes. The coupling was repeated once at 40 °C, with each reaction lasting 4 hours, to complete sequence coupling and obtain peptide resin. The lysis reagent was TFA:EDT:TIS:water (volume ratio) of 90:5:2.5:2.5. After lysis, MTBE precipitated, crystallized, and analyzed by solid-state HPLC, yielding a purity of 56.76%, lacking Ile. 23 Impurities: 18.47%.

[0086] Comparative Example 4: The amino acid equivalents of Fmoc-Ile-OH (position 23) were determined using 3 eq (5.31 g), PyBop 7.80 g, DIEA 2.13 g, and 60 mL of LDMF solution, activated in an ice bath for 5 minutes. The reaction was carried out at 40 °C for 24 h to complete sequence coupling, yielding peptide resin. The lysis reagent was TFA:EDT:TIS:water (volume ratio) of 90:5:2.5:2.5. After lysis, MTBE precipitated, crystallized, and analyzed by solid-state HPLC. The purity was 54.98%, with Ile missing. 23 Impurities: 18.84%.

[0087] The Fmoc-Ile-OH reaction was optimized as follows: 1. Different types of condensation were used, including DIC / OxymaPure, HATU / DIEA, PyBop / DIEA, DEPBT / DIEA, and TBTU / DIEA; 2. The reaction time was extended by 4 h, 8 h, and 24 h; 3. The reaction temperature was increased to 30℃, 40℃, and 50℃; 4. Repeated coupling and increasing the amino acid equivalent (3 eq, 6 eq) were employed. All these methods resulted in a residual starting material of over 15%, while the Fmoc-Ile-Glu(OtBu)-OH reaction avoided the Ile deficiency from the outset. 23 Impurities were removed by reacting with DIC / Oxyma Pure for 2-3 hours until the reactants were completely reacted.

[0088] Conclusion: Sequence Ile23 -Glu 24 Various synthetic methods were employed, including stepwise synthesis, where amino acids Fmoc-Glu(OtBu)-OH were coupled first, followed by coupling with Fmoc-Ile-OH, all of which resulted in the deletion of Ile. 23 Impurities >15%. After purification and lyophilization, the purity was greater than 98.5%, single impurity <0.2%, and the overall yield was 27%. This indicates that existing synthetic strategies struggle to control the formation of missing impurities, and even when high-purity products are obtained through purification, the yield remains too low. The method of this invention effectively avoids the formation of missing Ile impurities, resulting in a high-purity crude Retatrutide product that facilitates subsequent purification, significantly improving both product purity and overall yield.

[0089] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for synthesizing retatrutide, characterized in that, include: Peptide resins were prepared by sequential coupling according to the Retatrutide sequence; The peptide resin was cleaved to obtain Retatrutide; wherein Ile 23 -Glu 24 The coupling raw material for Glu is Fmoc-Glu(OtBu)-OH.

2. The synthesis method according to claim 1, characterized in that, The sequential coupling includes segmented coupling, and the coupling order is: Ser, Pro-Pro, Pro, Ala, Gly, Ser, Ser, Pro, Gly-Gly, Glu, Leu, Leu, Tyr, Ile-Glu, Phe, Ala, Aib, Gln, Ala, Lys[C20-γGlu-AEEA], Lys, Asp, Leu, α-Me-Leu, Ile, Ser, Tyr, Asp, Ser, Thr, Phe, Thr, Gly, Gln and Tyr-Aib.

3. The synthesis method according to claim 2, characterized in that, The Tyr 1 -Aib 2 The coupling raw materials are Boc-Tyr(tBu)-Aib-OH, Gln 3 The coupling raw materials are Fmoc-Gln(Trt)-OH and Gly 4 The coupling raw material is Fmoc-Gly-OH,Thr 5 The coupling raw materials are Fmoc-Thr(tBu)-OH and Phe 6 The coupling raw material is Fmoc-Phe-OH,Thr 7 The coupling raw materials are Fmoc-Thr(tBu)-OH and Ser 8 The coupling raw materials are Fmoc-Ser(tBu)-OH and Asp. 9 The coupling raw material is Fmoc-Asp(OtBu)-OH, Tyr 10 The coupling raw materials are Fmoc-Tyr(tBu)-OH and Ser 11 The coupling raw material is Fmoc-Ser(tBu)-OH, Ile 12 The coupling raw materials are Fmoc-Ile-OH and αMeLeu. 13 The coupling raw material is Fmoc-[α-Me-Leu]-OH, Leu 14 The coupling raw materials are Fmoc-Leu-OH and Asp. 15 The coupling raw materials are Fmoc-Asp(OtBu)-OH and Lys 16 The coupling raw material is Fmoc-Lys(Boc)-OH, Lys 17 The coupling agent for [C20-γGlu-AEEA] is Fmoc-Lys[C20-OtBu-γGlu(OtBu)-AEEA]-OH, Ala 18 The coupling raw materials are Fmoc-Ala-OH and Gln. 19 The coupling raw materials are Fmoc-Gln(Trt)-OH and Aib. 20 The coupling raw materials are Fmoc-Aib-OH and Ala 21 The coupling raw materials are Fmoc-Ala-OH and Phe 22 The coupling raw materials are Fmoc-Phe-OH and Ile. 23 -Glu 24 The coupling raw material is Fmoc-Ile-Glu(OtBu)-OH, Tyr 25 The coupling raw materials are Fmoc-Tyr(tBu)-OH and Leu 26 The coupling raw material is Fmoc-Leu-OH, Leu 27 The coupling raw materials are Fmoc-Leu-OH and Glu. 28 The coupling raw materials are Fmoc-Glu(OtBu)-OH and Gly 29 -Gly 30 The coupling raw material is Fmoc-Gly-Gly-OH, Pro 31 The coupling raw materials are Fmoc-Pro-OH and Ser 32 The coupling raw material is Fmoc-Ser(tBu)-OH, Ser 33 The coupling raw materials are Fmoc-Ser(tBu)-OH and Gly 34 The coupling raw materials are Fmoc-Gly-OH and Ala 35 The coupling raw material is Fmoc-Ala-OH, Pro 36 The coupling raw material is Fmoc-Pro-OH, Pro 37 -Pro 38 The coupling raw materials are Fmoc-Pro-Pro-OH and Ser 39 The coupling raw material is Fmoc-Ser(tBu)-OH.

4. The synthesis method according to claim 1, characterized in that, The resin is an amino resin, selected from one of RinkAmide AM Resin, Rink Amide MBHA Resin, or Sieber Resin. The resin has a crosslinking degree of 1% to 2%, a particle size of 100 to 200 mesh, and a substitution degree of 0.3 to 0.8 mmol / g.

5. The synthesis method according to claim 1, characterized in that, The amino acid equivalent of the coupling raw material is 2.0~3.0 eq.

6. The synthesis method according to claim 1, characterized in that, The coupling includes the steps of removing the N-terminal protecting group and condensation; The reagent for removing the N-terminal protecting group includes a mixed solution of piperidine and DMF, wherein the volume percentage of piperidine is 20% and the volume percentage of DMF is 80%, and the reaction time for removing the N-terminal protecting group is 15 min. The condensation reagent is selected from one or more of DIC / HOBt, DIC / Oxyma Pure, HBTU / DIEA or PyBop / DIEA, and the activation time of condensation is 5 min; The coupling reaction solvent is selected from one or more of DCM, DMF, NMP or DMSO, the reaction time is 2-3 h, and the reaction temperature is 20-30 °C.

7. The synthesis method according to claim 1, characterized in that, The reagents used in the peptide resin cleavage process include trifluoroacetic acid, 1,2-ethylenedithiol, triisopropylsilane, and water. The volume ratio of trifluoroacetic acid, 1,2-ethylenedithiol, triisopropylsilane and water is (80~100):(1~10):(0.5~5):(0.5~5), the reaction time is 2~3h, and the reaction temperature is 20~30℃.

8. The synthesis method according to claim 1, characterized in that, The synthesis method further includes a precipitation step, wherein the precipitating reagent includes anhydrous methyl tert-butyl ether.

9. The synthesis method according to claim 1, characterized in that, It also includes a purification step, wherein the purification conditions include: The chromatographic column is a C18 column; Mobile phase A is an acidic aqueous solution, and mobile phase B is an acetonitrile solution or an acidic acetonitrile solution; The acidic aqueous solution is selected from a trifluoroacetic acid aqueous solution with a volume percentage of 0.03% to 2.0%, or an acetic acid aqueous solution with a volume percentage of 0.3% to 5.0%. The acidic acetonitrile solution includes an acetic acid-acetonitrile solution, wherein the volume percentage of acetic acid is 0.5-1.5%.

10. The synthesis method according to claim 9, characterized in that, The elution method is gradient elution, and the specific elution procedure is as follows: From 0 to 10 minutes, the volume fraction of phase A was 90%, and the volume fraction of phase B was 10%. Over 10-15 minutes, the volume fraction of phase A decreased from 90% to 60%, and the volume fraction of phase B decreased from 10% to 40%. Over 15 to 80 minutes, the volume fraction of phase A decreased from 60% to 45%, and the volume fraction of phase B decreased from 40% to 55%.

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