Method for preparing tirzepatide

Abstract

A method for synthesizing tirzepatide, which method mainly comprises the following steps: 1) coupling an amino resin, which serves as a starting resin, with a protected amino acid and polypeptide fragments by means of a solid-phase synthesis method to obtain a peptide resin of tirzepatide, wherein a Glu-Gly dipeptide fragment is used for Glu3-Gly4, a Thr-Phe dipeptide fragment is used for Thr5-Phe6, a Lys[AEEA-AEEA-γ-Glu-C20] fragment is used for Lys20, and a Gly-Gly dipeptide fragment is used for Gly29-Gly30; 2) cleaving the peptide resin to obtain crude tirzepatide; and 3) purifying the crude tirzepatide by means of reversed-phase chromatography to obtain purified tirzepatide. The method can effectively reduce the generation of [D-Glu] racemic impurity, [D-Thr] racemic impurity, [D-Phe] racemic impurity and [+Gly] impurity, thereby improving the purity and yield of tirzepatide, significantly reducing the difficulty of purifying the crude tirzepatide, and substantially improving the overall yield of tirzepatide on the premise of ensuring the purity of tirzepatide. In addition, the synthesis of multiple fragments can proceed concurrently, thereby shortening the synthesis time. The purity of the prepared crude tirzepatide can reach 86.22% or higher, and the yield of the crude tirzepatide can reach 101.51% or higher. After a simple purification step, these impurities in tirzepatide are substantially removed. The purity of the purified tirzepatide can reach 99.21% or higher, with the maximum single impurity content being below 0.15%, and the overall yield can reach 63.02% or higher. The method reduces the synthesis costs and is conducive to large-scale industrial production.

Description

A method for preparing telpoeptide Technical Field

[0001] This invention belongs to the field of polypeptide drug preparation, specifically a method for preparing telpoeptide. Background Technology

[0002] Tirzepatide is a synthetic mimic of two hormones, GLP-1 and GIP, and can improve blood glucose levels and weight in adults with type 2 diabetes. The sequence structure of Tirzepatide is as follows:

[0003] Tyr 1 -Aib-Glu-Gly-Thr 5 -Phe-Thr-Ser-Asp-Tyr 10 -Ser-Ile-Aib-Leu-Asp 15 -Lys-Ile-Ala-Gln-Lys 20 (AEEA-AEEA-γGlu-Eicosanedioicacid)-Ala-Phe-Val-Gln-Trp 25 -Leu-Ile-Ala-Gly-Gly 30 -Pro-Ser-Ser-Gly-Ala 35 -Pro-Pro-Pro-Ser-NH2.

[0004] Telpotrioside has been approved by the FDA for the treatment of patients with poorly controlled type 2 diabetes. It is the first and only FDA-approved GIP and GLP receptor agonist, with a mechanism of action similar to GLP-1 receptor agonists but with the addition of GIP, thus being called a dual agonist. Furthermore, after oral administration, the intestines naturally release these two hormones, creating a feeling of fullness and aiding in weight loss.

[0005] Patent CN107207576A discloses a method for solid-phase preparation of Tirzepatide, which involves stepwise solid-phase synthesis of a 39-amino acid linear peptide, selective removal of the Lys side-chain protecting group Alloc, solid-phase coupling of side-chain modification groups, and cleavage to obtain the polypeptide product. This method suffers from numerous steps, a long cycle, many impurities, and difficult purification. Patent CN110903355A discloses another method for solid-phase preparation of Tirzepatide, also employing a stepwise coupling method. The insertion of amino acid 20 is selected from Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mono-tBu))-OH, and the insertion of amino acids 1-4 is selected from the tetrapeptide fragment Boc-Tyr(tBu)-Aib-Glu(tBu)-Gly-OH, followed by cleavage to obtain the polypeptide product. Although this method uses partial fragment coupling, the generation of related deletion peptide impurities and racemic peptide impurities is still unavoidable. Because Tirzepatide has a long sequence and contains many hydrophobic amino acids, it is prone to folding during synthesis using stepwise amino acid condensation, leading to severe resin shrinkage, prolonged reaction time, and the generation of numerous impurities in the crude peptide that are very similar to the product properties, such as racemic impurities, deleted peptides, and added amino acid peptides. Simultaneously, Tirzepatide contains multiple Aibs, which are difficult to couple due to steric hindrance, and coupling with adjacent amino acids is also challenging. On the one hand, these impurities have similar physicochemical properties to Tirzepatide, greatly increasing the difficulty of separating and purifying the product, resulting in a significantly reduced yield; on the other hand, the high impurity content reduces the purity and content of the target peptide, leading to a lower yield and significantly increased costs. Furthermore, impurity control is crucial for drug quality control. The types and amounts of impurities in a drug are collectively referred to as the impurity profile. Any substance that affects drug purity is collectively called an impurity. Adverse reactions in clinical use are sometimes related to impurities present in the drug, in addition to its pharmacological activity. Standardizing impurity research and controlling them within a safe and reasonable range is the core issue in controlling the drug impurity profile.

[0006] Therefore, there is an urgent need for a method to prepare telpoeptide with fewer types of impurities, lower impurity content, lower synthesis cost, and higher product purity and yield. Summary of the Invention

[0007] To overcome the difficulties in coupling, complex operation, and resin condensation encountered during the synthesis of telpoeptide, and to solve the problems of numerous impurities, high impurity content, low product purity, and low yield, this invention provides a method for preparing telpoeptide. This method can effectively reduce the generation of [D-Glu] racemic impurities, [D-Thr] racemic impurities, [D-Phe] racemic impurities, and [+Gly] impurities, thereby improving the purity and yield of telpoeptide and facilitating its large-scale production.

[0008] To achieve the objectives of this invention, the following technical solutions are provided:

[0009] A method for preparing telpoeptide, characterized by comprising the following main steps:

[0010] 1) Using amino resin as the starting resin, a solid-phase synthesis method is adopted to couple it with protected amino acids and polypeptide fragments to obtain the peptide resin of telpoeptide.

[0011] Among them, Glu3-Gly4 uses the Glu-Gly dipeptide fragment, Thr 5 -Phe 6 Using Thr-Phe dipeptide fragment, Lys 20 Using Lys[AEEA-AEEA-γ-Glu-C 20 [Excerpt, Gly] 29 -Gly 30 Using the Gly-Gly dipeptide fragment;

[0012] 2) Peptide resin cleavage yields crude telpoeptide peptide;

[0013] 3) The crude peptide was purified by reversed-phase chromatography to obtain the refined peptide, telpoeptide.

[0014] The applicant unexpectedly discovered that using the above fragments in the synthesis of telpoeptide can significantly inhibit / reduce the generation of [D-Glu] racemic impurities, [D-Thr] racemic impurities, [D-Phe] racemic impurities, and [+Gly] impurities, thereby significantly improving the yield and purity of telpoeptide.

[0015] Meanwhile, in the preparation of telpoeptide, the 20-position uses the monomer Lys[AEEA-AEEA-γ-Glu-C] 20 This fragment allows peptide intermediates to be easily inserted into SPPS, makes it easier for amino-terminal amino acids to react with them, and can significantly inhibit / reduce the generation of mismatched peptide impurities (such as amino acid-deficient peptides and amino acid-excess peptides) and racemic peptide impurities, thus significantly improving the yield and purity of telpoeptide.

[0016] [D-Glu] racemic impurities:

[0017] H-Tyr-Aib-D-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys(AEEA-AEEA-γGlu-Eicosanedioicacid)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;

[0018] [D-Thr] racemic impurity

[0019] H-Tyr-Aib-Glu-Gly-D-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys(AEEA-AEEA-γGlu-Eicosanedioicacid)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;

[0020] [D-Phe] racemic impurity:

[0021] H-Tyr-Aib-Glu-Gly-Thr-D-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys(AEEA-AEEA-γGlu-Eicosanedioicacid)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;

[0022] [+Gly] impurity:

[0023] H-Tyr-Aib-Glu-Gly-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys(AEEA-AEEA-γGlu-Eicosanedioicacid)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;

[0024] In some implementations, Ile12-Aib13 uses the Ile-Aib dipeptide fragment.

[0025] The applicant unexpectedly discovered that using the Ile-Aib dipeptide fragment could significantly inhibit / reduce the generation of [D-Ile] racemic impurities and [-Ile] deletion impurities.

[0026] [D-Ile] racemic impurities:

[0027] H-Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-D-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys(AEEA-AEEA-γGlu- Eicosanedioicacid)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;

[0028] [-Ile] Missing impurities:

[0029] H-Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys(AEEA-AEEA-γGlu-Eic osanedioicacid)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;

[0030] In some implementations, Tyr1-Aib2 uses the Tyr-Aib dipeptide fragment.

[0031] The applicant unexpectedly discovered that Tyr1-Aib2, using the Tyr-Aib dipeptide fragment, can significantly inhibit and reduce the generation of [D-Tyr] racemic impurities and [-Tyr] deletion impurities.

[0032] [D-Tyr] racemic impurities:

[0033] HD-Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys(AEEA-AEEA-γGlu- Eicosanedioicacid)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;

[0034] [-Tyr] Missing impurities

[0035] H-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys(AEEA-AEEA-γGlu-Eic osanedioicacid)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;

[0036] In some implementations, Tyr1-Aib2 uses the Tyr-Aib dipeptide fragment, and Ile12-Aib13 uses the Ile-Aib dipeptide fragment.

[0037] The applicant unexpectedly discovered that by adopting the above scheme, the generation of [D-Tyr] racemic impurities, [-Tyr] deletion impurities, [D-Glu] racemic impurities, [D-Thr] racemic impurities, [D-Phe] racemic impurities, [+Gly] impurities, [D-Ile] racemic impurities, and [-Ile] deletion impurities can be significantly suppressed / reduced simultaneously.

[0038] In some implementations, Ala35-Pro36 uses the Ala-Pro dipeptide fragment.

[0039] In some embodiments, the amino resin in step (1) is selected from one of Rink Amide AM Resin, Rink Amide MBHA Resin, Sieber resin, and PAL MBHA Resin.

[0040] In some embodiments, the degree of resin substitution is 0.3-0.6 mmol / g.

[0041] In some embodiments, the lysis reagent in step 2) of the lysis step contains more than 90% trifluoroacetic acid.

[0042] In some embodiments, the cleavage reagent further comprises other components selected from water, phenol, 3-mercaptopropionic acid, and triisopropylsilane, with each other component accounting for 1%-5%.

[0043] In some implementations, the Glu-Gly dipeptide fragment is Fmoc-Glu(OtBu)-Gly-OH.

[0044] In some implementations, the Thr-Phe dipeptide fragment is Fmoc-Thr(tBu)-Phe-OH.

[0045] In some implementations, Lys[AEEA-AEEA-γ-Glu-C 20 The fragment is Fmoc-Lys[AEEA-AEEA-γ-Glu(OtBu)-C 20 -OtBu]-OH.

[0046] In some implementations, the Gly-Gly dipeptide fragment is Fmoc-Gly-Gly-OH.

[0047] In some implementations, the Ile-Aib dipeptide fragment is Fmoc-Ile-Aib-OH.

[0048] In some implementations, the Tyr-Aib dipeptide fragment is selected from Boc-Tyr(tBu)-Aib-OH and Fmoc-Tyr(tBu)-Aib-OH.

[0049] Based on long-term experimental research and studies on amino acid racemization and double intercalation, the applicant unexpectedly discovered that when amino acids such as tyrosine (Tyr), 2-aminoisobutyric acid (Aib), glutamic acid (Glu), glycine (Gly), threonine (Thr), phenylalanine (Phe), and isoleucine (Ile) participate in the synthesis of telpoeptide as single amino acids, steric hindrance, double intercalation, and racemization occur. For example, due to the special structure of Aib and the inherent stereochemistry of telpoeptide amino acid residues, the steric hindrance at this site is large, making the coupling reaction difficult, incomplete, and time-consuming. Single coupling of Gly will result in double intercalation to generate +Gly impurities; single coupling of Tyr and Ile will result in deletion to generate deletion peptide impurities; single coupling of Tyr, Glu, Thr, Phe, and Ile requires active ester activation of the carboxyl terminus, which easily leads to racemization to generate racemic impurities. The sites where these impurities are generated are no longer coiled, making them very similar to the chemical and spatial structures of telpoeptide, making separation extremely difficult. Obtaining high-purity telpoeptide inevitably comes at the cost of significant yield loss. At the same time, the reduced impurity content improves the purity and content of the target peptide, resulting in a higher yield and significantly reduced costs.

[0050] This invention employs a solid-phase synthesis method combining fragmentation and stepwise synthesis to prepare multiple peptide fragments, which are then used as key starting materials in the solid-phase synthesis of telpoeptide. This method significantly and simultaneously inhibits / reduces the generation of [D-Tyr] racemic impurities, [-Tyr] deletion impurities, [D-Glu] racemic impurities, [D-Thr] racemic impurities, [D-Phe] racemic impurities, [+Gly] impurities, [D-Ile] racemic impurities, and [-Ile] deletion impurities. The highest content of [D-Tyr] racemic impurity is 0.12%, the highest content of [-Tyr] deletion impurity is 0.69%, the highest content of [D-Glu] racemic impurity is 0.09%, the highest content of [D-Thr] racemic impurity is 0.21%, the highest content of [D-Phe] racemic impurity is 0.37%, the highest content of [+Gly] impurity is 0.26%, the highest content of [D-Ile] racemic impurity is 0.63%, and the highest content of [-Ile] deletion impurity is 0.76%. This significantly reduces the difficulty of crude peptide purification and greatly improves the purity and yield of telpoeptide. The crude peptide purity prepared using the method of this invention can reach over 86.22%, and the crude peptide yield is over 101.51%. After a simple purification process, these impurities in telpoeptide have been largely removed, and the purity of the peptide can reach over 99.21%, with the largest single impurity being less than 0.15%, and the total yield reaching over 63.02%. This reduces the synthesis cost and is beneficial for large-scale industrial production. Attached Figure Description

[0051] Figure 1 is an HPLC chromatogram of the crude telpoide obtained in Example 1 of the present invention.

[0052] Figure 2 is an HPLC chromatogram of the telpoide peptide prepared in Example 1 of the present invention.

[0053] Figure 3 is an HPLC chromatogram of the crude telpoide obtained in Example 2 of the present invention.

[0054] Figure 4 is an HPLC chromatogram of the crude telpoeptide obtained in Example 3 of this invention.

[0055] Figure 5 is an HPLC chromatogram of the crude telpoide obtained in Example 4 of the present invention.

[0056] Figure 6 is an HPLC chromatogram of the crude telpoide obtained in Comparative Example 1 of this invention.

[0057] Figure 7 is an HPLC chromatogram of the telpoide peptide prepared in Comparative Example 1 of this invention. Detailed Implementation

[0058] The present invention will be further described in detail below through embodiments, which are intended to illustrate the invention and not limit it. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications also fall within the protection scope of the present invention.

[0059] Protecting groups are commonly used in amino acid synthesis to protect amino and carboxyl groups (such as amino and carboxyl groups) on the main chain and side chains of amino acids, preventing them from reacting and generating impurities during the preparation of the target product. For the amino acids requiring side chain protection in this invention, those skilled in the art are familiar with their side chain structures and know how to use common protecting groups to protect the amino and carboxyl groups on the amino acid side chains. Preferably, this invention protects the side chains of Ser, Tyr, and Thr with a tBu protecting group; the side chains of Trp and Lys with a Boc protecting group; the side chain of Gln with a Trt protecting group; and the side chains of Glu and Asp with an OtBu protecting group. Furthermore, in the amino acids involved in the method described in this invention, the N-terminus of the amino acids is preferably protected with an Fmoc protecting group, while Tyr can also be protected with a Boc protecting group. Depending on the synthetic requirements, the amino acids or peptides used in this invention, especially dipeptides, can be protected with protecting groups.

[0060] In this invention, the peptide fragments may have or may not have protecting groups, and may be obtained through commissioned synthesis (purchase) or synthesized using known methods.

[0061] Example 1: Preparation of telpolide 1

[0062] 1. Preparation of telpopeptide resin

[0063] Weigh 33.33 g (10 mmol) of Rink Amide AM Resin with a degree of substitution of 0.30 mmol / g and add it to a solid-phase reaction column. Wash once with DMF, swell the Rink Amide AM Resin with DMF for 30 minutes, remove Fmoc protection with a DMF:piperidine mixture of 4:1 (v / v), and then wash six times with DMF. Weigh 11.50 g of Fmoc-Ser(tBu)-OH and 4.46 g of HOBt, dissolve them in DMF solution, cool to below 5°C, add 5.68 g of DIC for activation, and then add the solution to the resin-packed reaction column. React at room temperature for 2 hours. Use ninhydrin to determine the reaction endpoint. If the resin is colorless and transparent, the reaction is complete; if the resin is colored, the reaction is incomplete and needs to be continued for another hour, or the same amount of reagent should be added again, or the condensation reagent should be replaced and added again. Ninhydrin detection is applicable to subsequent amino acid coupling reactions to determine the reaction endpoint. Repeat the steps above to remove Fmoc protection and add the corresponding amino acid conjugation, and sequentially insert the protective amino acids or peptide fragments as shown in Table 1 according to the telpoide sequence:

[0064] Table 1

[0065] The resulting thiopeptide resin is Boc-Tyr(tBu)-{Aib}-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-{Aib}-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys{diacid-C 20 The product, γGlu(OtBu)-AEEA-AEEA}-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin, weighed 95.56 g. No peptide resin volume shrinkage occurred during the coupling process, indicating easy coupling. Each reaction lasted 1.5 h. The resin was colorless and transparent as determined by the ninhydrin method.

[0066] 2. Preparation of crude telpoeptide

[0067] 95.56 g of telpoeptide peptide resin was added to a 2000 ml three-necked round-bottom flask. 960 ml of lysis buffer was prepared using a TFA:Tis:Mpr:H2O ratio of 93.5:0.5:3.0:3.0 (v / v). After thorough mixing, the above peptide resin was added, and the mixture was reacted at room temperature for 3 hours. The mixture was filtered, and the filtrate was added to 8 times its volume of ice-cold isopropyl ether for precipitation for 1 hour. The filtrate was centrifuged, washed four times with isopropyl ether, and dried to obtain 49.34 g of crude telpoeptide peptide as a white solid. The purity of the crude peptide was measured to be 88.78%, and the yield was 102.50%. The HPLC chromatogram and data of the crude telpoeptide peptide are shown in Figure 1 and Table 2.

[0068] Table 2

[0069] 3. Purification of crude telpoeptide

[0070] A preparative liquid chromatography system (RP-PLC) was used with a wavelength of 220 nm. The chromatographic column was a reversed-phase C8 column, with 10 mM ammonium bicarbonate aqueous solution as mobile phase A and acetonitrile as mobile phase B. After RP-PLC purification, salt transfer was performed, the target peptide fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain telpoide peptide. The purity of telpoide peptide was determined to be 99.77%, the maximum single impurity was 0.10%, and the overall yield was 63.39%. The chromatograms of telpoide peptide obtained using this invention are shown in Figure 2 and Table 3.

[0071] Table 3

[0072] Example 2: Preparation of telpoeptide 2

[0073] 1. Preparation of telpopeptide resin

[0074] Weigh 16.67 g (10 mmol) of Rink Amide MBHA Resin with a degree of substitution of 0.60 mmol / g and add it to a solid-phase reaction column. Wash once with DMF, swell the Rink Amide MBHA Resin with DMF for 30 minutes, remove Fmoc protection with a DMF:piperidine mixture of 4:1 (v / v), and then wash 6 times with DMF. Weigh 11.50 g of Fmoc-Ser(tBu)-OH and 4.46 g of HOBt and dissolve them in DMF solution. After cooling to below 5°C, add 5.68 g of DIC for activation and then add it to the above-mentioned reaction column packed with resin. After reacting at room temperature for 2 hours, use ninhydrin to determine the reaction endpoint. If the resin is colorless and transparent, the reaction is complete; if the resin is colored, the reaction is incomplete and needs to be continued for another hour, or a single dose should be added again, or a different condensation reagent should be used for re-addition. Ninhydrin detection is applicable to subsequent amino acid coupling reactions to determine the reaction endpoint. Repeat the steps above to remove Fmoc protection and add the corresponding amino acid conjugation, and sequentially insert the protective amino acids or peptide fragments as shown in Table 4 according to the telpoide sequence:

[0075] Table 4

[0076] The resulting thiopeptide resin is Boc-Tyr(tBu)-{Aib}-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-{Aib}-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys{diacid-C 20 The product, γGlu(OtBu)-AEEA-AEEA}-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide MBHA Resin, weighed 78.11 g. No peptide resin volume shrinkage occurred during the coupling process, indicating easy coupling. Each reaction lasted 1.5 h. The resin was colorless and transparent as determined by the ninhydrin method.

[0077] 2. Preparation of crude telpoeptide

[0078] 78.11 g of telpoeptide resin was added to a 2000 ml three-necked round-bottom flask. 780 ml of lysis buffer was prepared using a TFA:Tis:Mpr:H2O ratio of 93.5:0.5:3.0:3.0 (v / v). After thorough mixing, the above-mentioned peptide resin was added, and the mixture was reacted at room temperature for 3 hours. The mixture was filtered, and the filtrate was added to 8 times its volume of ice-cold isopropyl ether for precipitation for 1 hour. The filtrate was centrifuged, washed four times with isopropyl ether, and dried to obtain 48.86 g of crude telpoeptide as a white solid. The purity of the crude peptide was measured to be 86.22%, and the yield was 101.51%. The chromatogram and data of the crude telpoeptide are shown in Figure 3 and Table 5.

[0079] Table 5

[0080] 3. Purification of crude telpoeptide

[0081] A preparative liquid chromatography system (RP-PLC) was used with a wavelength of 220 nm. The column was a reversed-phase C8 column, with 10 mM ammonium bicarbonate aqueous solution as mobile phase A and acetonitrile as mobile phase B. After RP-PLC purification, salt transfer was performed, the target peptide fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain telpoide peptide. The purity of telpoide peptide was determined to be 99.21%, the maximum single impurity was 0.13%, and the overall yield was 64.32%. The chromatogram of telpoide peptide obtained using this invention is similar to that shown in Figure 2.

[0082] Example 3: Preparation of telpoeptide 3

[0083] 1. Preparation of telpopeptide resin

[0084] Weigh 33.33 g (10 mmol) of Sieber Amide Resin with a degree of substitution of 0.30 mmol / g and add it to a solid-phase reaction column. Wash once with DMF, swell the Sieber Amide Resin with DMF for 30 minutes, remove the Fmoc protection with a DMF:piperidine mixture of 4:1 (v / v), and then wash six times with DMF. Weigh 11.50 g of Fmoc-Ser(tBu)-OH and 4.46 g of HOBt and dissolve them in DMF solution. After cooling to below 5°C, add 5.68 g of DIC for activation and then add it to the above-mentioned reaction column packed with resin. After reacting at room temperature for 2 hours, use ninhydrin to determine the reaction endpoint. If the resin is colorless and transparent, the reaction is complete; if the resin is colored, the reaction is incomplete and needs to be continued for another hour, or the amount of reagent should be repeated, or a different condensation reagent should be used for repeated reactions. Ninhydrin detection is applicable to subsequent amino acid coupling reactions to determine the reaction endpoint. Repeat the steps above to remove Fmoc protection and add the corresponding amino acid conjugation, and sequentially insert the protective amino acids or peptide fragments as shown in Table 6 according to the telpoide sequence:

[0085] Table 6

[0086] The resulting thiopeptide resin is Boc-Tyr(tBu)-{Aib}-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-{Aib}-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys{diacid-C 20 The sample was γGlu(OtBu)-AEEA-AEEA}-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Sieber Amide Resin, weighing 95.11g. No peptide resin volume shrinkage occurred during coupling, indicating easy coupling. Each reaction lasted 1.5 hours. The resin was colorless and transparent when tested using the ninhydrin method.

[0087] 2. Preparation of crude telpoeptide

[0088] 95.11 g of telpoeptide resin was added to a 2000 ml three-necked round-bottom flask. 950 ml of lysis buffer was prepared using a TFA:Tis:Mpr:H2O ratio of 93.5:0.5:3.0:3.0 (v / v). After thorough mixing, the above-mentioned peptide resin was added, and the mixture was reacted at room temperature for 3 hours. The mixture was filtered, and the filtrate was added to 8 times its volume of ice-cold isopropyl ether for precipitation for 1 hour. The filtrate was centrifuged, washed four times with isopropyl ether, and dried to obtain 50.32 g of crude telpoeptide as a white solid. The purity of the crude peptide was measured to be 87.39%, and the yield was 104.55%. The chromatogram and data of the crude telpoeptide are shown in Figure 4 and Table 7.

[0089] Table 7

[0090] 3. Purification of crude telpoeptide

[0091] The crude telpoeptide obtained in Embodiment 3 of this invention was processed using an RP-PLC preparative liquid chromatography system at a wavelength of 220 nm. The chromatographic column was a reversed-phase C8 column, with 10 mM ammonium bicarbonate aqueous solution as mobile phase A and acetonitrile as mobile phase B. After RP-PLC purification, salt transfer was performed, the target peptide fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain refined telpoeptide. The purity of the refined telpoeptide was determined to be 99.30%, the maximum single impurity was 0.11%, and the total yield was 65.46%. The chromatogram of the refined telpoeptide obtained using this invention is similar to that shown in Figure 2.

[0092] Example 4: Preparation method of telpoeptide 4

[0093] 1. Preparation of telpopeptide resin

[0094] Weigh 33.33 g (10 mmol) of PAL MBHA Resin with a degree of substitution of 0.30 mmol / g and add it to a solid-phase reaction column. Wash once with DMF, swell the PAL MBHA Resin with DMF for 30 minutes, remove Fmoc protection with a DMF:piperidine mixture of 4:1 (v / v), and then wash six times with DMF. Weigh 11.50 g of Fmoc-Ser(tBu)-OH and 4.46 g of HOBt and dissolve them in DMF solution. After cooling to below 5°C, add 5.68 g of DIC for activation and then add it to the above-mentioned reaction column packed with resin. After reacting at room temperature for 2 hours, use ninhydrin to determine the reaction endpoint. If the resin is colorless and transparent, the reaction is complete; if the resin is colored, the reaction is incomplete and needs to be continued for another hour, or the amount of reagent should be repeated, or the condensation reagent should be replaced and repeated. Ninhydrin detection is applicable to subsequent amino acid coupling reactions to determine the reaction endpoint. Repeat the steps above to remove Fmoc protection and add the corresponding amino acid conjugation, and sequentially insert the protective amino acids or peptide fragments as shown in Table 8 according to the telpoide sequence:

[0095] Table 8

[0096] The resulting thiopeptide resin is Boc-Tyr(tBu)-{Aib}-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-{Aib}-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys{diacid-C 20The product, γGlu(OtBu)-AEEA-AEEA}-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-PAL MBHA Resin, weighed 95.05g. No peptide resin volume shrinkage occurred during the coupling process, indicating easy coupling. Each reaction lasted 1.5 hours. The resin was colorless and transparent when tested using the ninhydrin method.

[0097] 2. Preparation of crude telpoeptide

[0098] 95.05 g of telpopeptide resin was added to a 2000 ml three-necked round-bottom flask. 950 ml of lysis buffer was prepared using a TFA:Tis:Mpr:H2O ratio of 93.5:0.5:3.0:3.0 (v / v). After thorough mixing, the above-mentioned peptide resin was added, and the mixture was reacted at room temperature for 3 hours. The mixture was filtered, and the filtrate was added to 8 times its volume of ice-cold isopropyl ether for precipitation for 1 hour. The filtrate was centrifuged, washed four times with isopropyl ether, and dried to obtain 49.82 g of white solid telpopeptide crude peptide. The purity of the crude peptide was measured to be 88.49%, and the yield was 103.51%. The chromatogram and data of the crude telpopeptide are shown in Figure 5 and Table 9.

[0099] Table 9

[0100] 3. Purification of crude telpoeptide 4

[0101] A preparative liquid chromatography system (RP-PLC) was used with a wavelength of 220 nm. The chromatographic column was a reversed-phase C8 column, with 10 mM ammonium bicarbonate aqueous solution as mobile phase A and acetonitrile as mobile phase B. After RP-PLC purification, salt transfer was performed, the target peptide fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain telpoide peptide. The purity of telpoide peptide was determined to be 99.47%, the maximum single impurity was 0.15%, and the overall yield was 63.02%. The chromatogram of telpoide peptide prepared using this invention is similar to that shown in Figure 2.

[0102] This invention solves the problems of increasingly difficult subsequent amino acid coupling, numerous types of impurities, and high impurity content in the synthesis of telpoeptide using conventional solid-phase methods, thereby improving the purity and yield of the crude peptide. The predicted molecular weight of telpoeptide is 4810.5200. Mass spectrometry analysis confirmed that all telpoeptides prepared by this invention have a molecular weight of 4810.5245, meeting the expected result and proving the successful preparation of telpoeptide by this invention.

[0103] Comparative Example 1:

[0104] 1. Preparation of telpopeptide resin

[0105] Weigh 33.33 g (10 mmol) of Rink Amide AM Resin with a degree of substitution of 0.30 mmol / g and add it to a solid-phase reaction column. Wash once with DMF, swell the Rink Amide AM Resin with DMF for 30 minutes, remove Fmoc protection with a DMF:piperidine mixture of 4:1 (v / v), and then wash six times with DMF. Weigh 11.50 g of Fmoc-Ser(tBu)-OH and 4.46 g of HOBt, dissolve them in DMF solution, cool to below 5°C, add 5.68 g of DIC for activation, and then add the solution to the resin-packed reaction column. React at room temperature for 2 hours. Use ninhydrin to determine the reaction endpoint. If the resin is colorless and transparent, the reaction is complete; if the resin is colored, the reaction is incomplete and needs to be continued for another hour, or the same amount of reagent should be added again, or the condensation reagent should be replaced and added again. Ninhydrin detection is applicable to subsequent amino acid coupling reactions to determine the reaction endpoint. Repeat the steps above to remove Fmoc protection and add the corresponding amino acid conjugation, and sequentially insert the protective amino acids or peptide fragments as shown in Table 10 according to the telpoide sequence:

[0106] Table 10

[0107] The resulting thiopeptide resin is Boc-Tyr(tBu)-{Aib}-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-{Aib}-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys{diacid-C 20 The product, γGlu(OtBu)-AEEA-AEEA}-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin, weighed 90.56 g. No peptide resin volume shrinkage occurred during the coupling process, indicating easy coupling. Each reaction lasted 1.5 h. The resin was colorless and transparent as determined by the ninhydrin method.

[0108] 2. Preparation of crude telpoeptide

[0109] 90.56 g of telpopeptide resin was added to a 2000 ml three-necked round-bottom flask. 910 ml of lysis buffer was prepared using a TFA:Tis:Mpr:H2O ratio of 93.5:0.5:3.0:3.0 (v / v). After thorough mixing, the above-mentioned peptide resin was added, and the mixture was reacted at room temperature for 3 hours. The mixture was filtered, and the filtrate was added to 8 times its volume of ice-cold isopropyl ether for precipitation for 1 hour. The filtrate was centrifuged, washed four times with isopropyl ether, and dried to obtain 45.34 g of white solid telpopeptide crude peptide. The purity of the crude peptide was measured to be 67.99%, and the yield was 94.20%. The chromatogram and data of the crude telpopeptide are shown in Figure 6 and Table 11.

[0110] Table 11

[0111] 3. Purification of crude telpoeptide

[0112] A preparative liquid chromatography system (RP-PLC) was used with a wavelength of 220 nm. The column was a reversed-phase C8 column, with 10 mM ammonium bicarbonate aqueous solution as mobile phase A and acetonitrile as mobile phase B. After RP-PLC purification, salt transfer was performed, the target peptide fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain telpoide. The purity of telpoide was determined to be 98.66%, the maximum single impurity was 0.22%, and the overall yield was 43.32%. The chromatogram and data of the crude telpoide are shown in Figure 7 and Table 12.

[0113] Table 12

[0114] As can be seen from the HPLC chromatograms and data in Examples 1-4 and Comparative Example 1, the synthesis method of the present invention greatly suppresses / reduces the generation of [D-Tyr] racemic impurities, [-Tyr] deletion impurities, [D-Glu] racemic impurities, [D-Thr] racemic impurities, [D-Phe] racemic impurities, [+Gly] impurities, [D-Ile] racemic impurities, and [-Ile] deletion impurities. The highest content of [D-Tyr] racemic impurity is 0.12%, the highest content of [-Tyr] deletion impurity is 0.69%, the highest content of [D-Glu] racemic impurity is 0.09%, the highest content of [D-Thr] racemic impurity is 0.21%, the highest content of [D-Phe] racemic impurity is 0.37%, the highest content of [+Gly] impurity is 0.26%, the highest content of [D-Ile] racemic impurity is 0.63%, and the highest content of [-Ile] deletion impurity is 0.76%. This significantly reduces the difficulty of crude peptide purification and greatly improves the purity and yield of telpoeptide. The crude peptide purity prepared using the method of this invention can reach over 86.22%, and the crude peptide yield is over 101.51%. After a simple purification process, these impurities in telpoeptide have been largely removed, and the purity of the peptide can reach over 99.21%, with the largest single impurity being less than 0.15%, and the total yield reaching over 63.02%. In the comparative examples, the content of [D-Tyr] racemic impurity was 1.24%, [-Tyr] deletion impurity was 0.23%, [D-Glu] racemic impurity was 5.61%, [D-Thr] racemic impurity was 1.78%, [D-Phe] racemic impurity was 3.81%, [+Gly] impurity was 4.1%, [D-Ile] racemic impurity was 2.31%, and [-Ile] deletion impurity was 1.66%, all of which were significantly higher than those in the method of this invention. Furthermore, the figure shows that the variety of impurities was also significantly greater than in the method of this invention. The crude peptide purity was 67.99%, and the crude peptide yield was 94.20%, both far lower than those in the method of this invention. The purified peptide purity was only 98.66%, with a maximum single impurity of 0.22% and a total yield of only 43.32%, far inferior to the method of this invention.

[0115] Therefore, the method of the present invention significantly reduces the generation of [D-Tyr] racemic impurities, [-Tyr] deletion impurities, [D-Glu] racemic impurities, [D-Thr] racemic impurities, [D-Phe] racemic impurities, [+Gly] impurities, [D-Ile] racemic impurities, and [-Ile] deletion impurities during the synthesis of telpolide. Simultaneously, it ensures the yield of telpolide, which is beneficial for purifying and obtaining high-purity, high-yield telpolide peptides.

[0116] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made 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. [Corrected according to Rule 91, 06.01.2025] A method for preparing telpolide, characterized in that, The main steps include: 1) Using amino resin as the starting resin, a solid-phase synthesis method is adopted to couple it with protected amino acids and polypeptide fragments to obtain the peptide resin of telpoeptide. Among them, Glu3-Gly4 uses the Glu-Gly dipeptide fragment, Thr 5 -Phe 6 Using Thr-Phe dipeptide fragment, Lys 20 Using Lys[AEEA-AEEA-γ-Glu-C 20 [Excerpt, Gly] 29 -Gly 30 Using the Gly-Gly dipeptide fragment; 2) Peptide resin cleavage yields crude telpoeptide peptide; 3) The crude peptide was purified by reversed-phase chromatography to obtain the refined peptide, telpoeptide.

2. The preparation method according to claim 1, characterized in that, Ile12-Aib13 uses the Ile-Aib dipeptide fragment.

3. The preparation method according to claim 1, characterized in that, Tyr1-Aib2 uses the Tyr-Aib dipeptide fragment.

4. The preparation method according to any one of claims 1-3, characterized in that, Tyr1-Aib2 uses the Tyr-Aib dipeptide fragment, and Ile12-Aib13 uses the Ile-Aib dipeptide fragment.

5. The preparation method according to claim 1, characterized in that, Ala35-Pro36 uses the Ala-Pro dipeptide fragment.

6. The preparation method according to claim 1, characterized in that, In step (1), the amino resin is selected from one of Rink Amide AM Resin, Rink Amide MBHA Resin, Sieber resin, and PAL MBHA Resin.

7. The preparation method according to claim 6, characterized in that, The degree of resin substitution is 0.3-0.6 mmol / g.

8. The preparation method according to claim 1, characterized in that, In step 2), the lysis reagent contains more than 90% trifluoroacetic acid.

9. The preparation method according to claim 8, characterized in that, The lysis reagent also contains other components selected from water, phenol, 3-mercaptopropionic acid, and triisopropylsilane, with each component accounting for 1%-5%.

10. The preparation method according to claim 1, characterized in that, The Glu-Gly dipeptide fragment is Fmoc-Glu(OtBu)-Gly-OH.

11. The preparation method according to claim 1, characterized in that, The Thr-Phe dipeptide fragment is Fmoc-Thr(tBu)-Phe-OH.

12. The preparation method according to claim 1, characterized in that, Lys[AEEA - AEEA - γ - Glu - C 20 fragment is Fmoc - Lys[AEEA - AEEA - γ - Glu(OtBu) - C 20 -OtBu]-OH.

13. The preparation method according to claim 1, characterized in that, The Gly-Gly dipeptide fragment is Fmoc-Gly-Gly-OH.

14. The preparation method according to claim 2, characterized in that, The Ile-Aib dipeptide fragment is Fmoc-Ile-Aib-OH.

15. The preparation method according to claim 3, characterized in that, The Tyr-Aib dipeptide fragment was selected from Boc-Tyr(tBu)-Aib-OH and Fmoc-Tyr(tBu)-Aib-OH.

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