A process for the preparation of tirzepatide
By using a combination of 5-7 peptide fragments and dipeptide fragments of Phe-Val-Gln-Trp-Leu in the preparation of Tirzepatide, the problems of high impurity content, difficult purification, and low yield in the existing technology were solved, and higher purification efficiency and yield were achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN JYMED TECH
- Filing Date
- 2021-04-02
- Publication Date
- 2026-06-16
AI Technical Summary
Existing methods for preparing Tirzepatide suffer from problems such as numerous impurities, difficulty in purification, and low yield. In particular, deletion peptides and racemic impurities are easily generated during stepwise amino acid coupling, which affects the purification difficulty and yield.
A solid-phase peptide synthesis method was adopted, which reduced the generation of missing peptides and racemic impurities and improved the yield by incorporating 5-7 peptide fragments of Phe-Val-Gln-Trp-Leu and combinations of dipeptide fragments such as Thr-Phe, Leu-Asp, Gly-Gly, and Ser-Ser.
It effectively reduced the generation of missing peptides and racemic impurities, lowered the purification difficulty, and improved the yield of Tirzepatide.
Smart Images
Figure BDA0003004885430000031
Abstract
Description
Technical Field
[0001] This invention relates to the field of polypeptide drug preparation, and specifically to a method for preparing Tirzepatide. Background Technology
[0002] Tirzepatide, a drug with dual receptor agonist activity targeting both glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1), is a key investigational product of Eli Lilly and Company. Currently undergoing Phase III clinical trials, it is expected to be approved for market launch in 2022. It is considered the most likely GLP-1 analogue hypoglycemic agent to challenge semaglutide's market dominance. Its peptide sequence is as follows: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Ile 17 -Ala 18 -Gln 19 -Lys 20 (AEEA-AEEA-γ-Glu-Eicosanedioic acid)-Ala 21 -Phe 22 -Val 23 -Gln 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -NH2.
[0003] Methods for preparing tirzepatide have been reported. CN107207576A discloses a solid-phase preparation method for tirzepatide, which involves the stepwise solid-phase synthesis of a 39-amino acid linear peptide and selective removal of Lys. 20 The method of using an Alloc side-chain protecting group for solid-phase coupling of side-chain modified groups followed by cleavage to obtain the polypeptide product is complex, involving numerous steps, a long cycle, many impurities, and difficult purification. CN110903355A discloses a solid-phase method for preparing Tirzepatide, which also employs a stepwise coupling method, wherein Lys... 20 The side chain is inserted using Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mono-tBu))-OH, and the amino acids at positions 1-4 are inserted using the tetrapeptide fragment Boc-Tyr(tBu)-Aib-Glu(tBu)-Gly-OH. Cleavage yields the polypeptide product. Although this method uses partial fragment coupling, the generation of related deletion peptides and racemic peptide impurities is still unavoidable. WO2020159949 discloses a solid-liquid combined method for preparing Tirzepatide, which first synthesizes fragments of different lengths in the solid phase, then condenses the fragments in the liquid phase to obtain the fully protected peptide, and finally cleaves it to obtain the polypeptide product. This method can solve some problems such as purification difficulties caused by related deletion peptide impurities, but the liquid-phase reaction is difficult to control, and the intermediates are difficult to purify, which is not conducive to large-scale industrial production. Therefore, providing a method for preparing Tirzepatide has important practical significance. Summary of the Invention
[0004] To address the problems of numerous impurities, difficult purification, and low yield in existing technologies, this invention provides a method for preparing Tirzepatide, comprising the following steps: preparing Tirzepatide peptide resin using a solid-phase polypeptide synthesis method, and obtaining Tirzepatide by cleavage of the Tirzepatide peptide resin; wherein the method for incorporating Phe-Val-Gln-Trp-Leu is as follows: using a 5-7 peptide fragment containing Phe-Val-Gln-Trp-Leu.
[0005] Preferably, the 5-7 peptide fragment containing Phe-Val-Gln-Trp-Leu is selected from:
[0006] Phe-Val-Gln-Trp-Leu-Ile-Ala;
[0007] Ala-Phe-Val-Gln-Trp-Leu-Ile;
[0008] Lys-Ala-Phe-Val-Gln-Trp-Leu.
[0009] Preferably, the 5-7 peptide fragment of Phe-Val-Gln-Trp-Leu is selected from:
[0010] Phe-Val-Gln-Trp-Leu-Ile;
[0011] Ala-Phe-Val-Gln-Trp-Leu.
[0012] As a preferred option, the 5-7 peptide fragment of Phe-Val-Gln-Trp-Leu is: Phe-Val-Gln-Trp-Leu.
[0013] In the above preparation method, the lysine at position 20 is introduced using either Fmoc-Lys(Alloc)-OH or Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH. When Fmoc-Lys(Alloc)-OH is used for lysine, the Tirzepatide main chain peptide resin is selectively deprotected by the Alloc protecting group, and then modified with side chains to obtain Tirzepatide peptide resin, which is then cleaved to obtain Tirzepatide. When Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH is used for lysine, Tirzepatide peptide resin can be directly synthesized, and then cleaved to obtain Tirzepatide.
[0014] Preferably, dipeptide fragments or combinations thereof are used in the preparation of Tirzepatide; wherein the dipeptide fragments are selected from Thr-Phe, Leu-Asp, Gly-Gly, and Ser-Ser. If individual amino acids or amino acid derivatives in the peptide backbone are coupled stepwise, it is difficult to sequentially couple Thr-Phe, Leu-Asp, Gly-Gly, and Ser-Ser in the peptide sequence as single amino acids, which easily leads to the formation of deletion or insertion peptides, thus affecting the yield of Tirzepatide. Coupling with Thr-Phe, Leu-Asp, Gly-Gly, and Ser-Ser dipeptide fragments or combinations thereof can effectively reduce the formation of deletion or insertion peptides, further improving the yield of Tirzepatide.
[0015] More preferably, in the preparation of Tirzepatide, a combination of a 5-7 peptide fragment and a dipeptide fragment containing Phe-Val-Gln-Trp-Leu is used, wherein the combination is as follows:
[0016] Phe-Val-Gln-Trp-Leu-Ile-Ala and Gly-Gly;
[0017] Lys-Ala-Phe-Val-Gln-Trp-Leu and Thr-Phe, Ser-Ser;
[0018] Phe-Val-Gln-Trp-Leu-Ile and Leu-Asp;
[0019] Phe-Val-Gln-Trp-Leu and Leu-Asp, Gly-Gly;
[0020] When synthesizing Tirzepatide using a fragment-coupled solid-phase synthesis method, only suitable fragments can suppress or reduce the generation of impurities such as deletion peptides, mismatched peptides, and racemic peptides. During the experimental process, the applicant discovered that during stepwise coupling to prepare Tirzepatide, the presence of numerous hydrophobic amino acids in the Tirzepatide sequence creates significant steric hindrance during stepwise coupling, leading to difficulties in the coupling of subsequent amino acids, especially Phe... 22 Val 23 Trp 25 and Leu 26 These four amino acids are more difficult to couple, and are prone to producing related deletion peptide impurities. Among them, the phenylalanine residue is also prone to racemization due to its own structure, forming D-Phe 22 Racemic byproducts. These impurities have properties similar to the product, and their increased content can affect purification difficulty and yield. Using the 5-7 peptide fragment of Phe-Val-Gln-Trp-Leu can not only reduce the production of peptides with single or multiple amino acid residue deletions, but also reduce D-Phe... 22 The generation of racemic byproducts reduces purification difficulty and increases yield. Furthermore, using Thr-Phe, Leu-Asp, Gly-Gly, Ser-Ser dipeptide fragments or combinations thereof can also reduce the generation of related deleted or inserted peptide impurities, facilitating purification.
[0021] This invention provides a method for preparing Tirzepatide by selecting a 5-7 peptide fragment containing Phe-Val-Gln-Trp-Leu for solid-phase coupling, which can reduce the generation of missing peptides and related racemic impurities, reduce purification difficulty, and improve yield. Detailed Implementation
[0022] The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to further understand the present invention. These embodiments should not be construed as limiting the scope of protection.
[0023] The Chinese names corresponding to the English abbreviations involved in this invention are shown in Table 1:
[0024] Table 1 shows the Chinese names corresponding to the English abbreviations involved in this invention.
[0025]
[0026] The technical solution of the present invention will be further described in detail according to the following embodiments.
[0027] Example 1: Preparation of Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-OH
[0028] (1) Preparation of Fmoc-Ala-2-CTC resin
[0029] 90.91 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 100 mmol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and washed three times. Fmoc-Ala-OH (62.23 g, 200 mmol), DIEA (51.70 g, 400 mmol), and 1.5 L of DMF were added, and the reaction was carried out for 6 h. The mixture was dried under vacuum, and methanol (9.61 g, 300 mmol), DIEA (12.93 g, 100 mmol), and 1.5 L of DMF were added. The reaction was carried out for 1 h, dried under vacuum, and washed three times each with DMF and DCM. The Fmoc-Ala-2-CTC resin was removed, dried, and the substitution degree of the Fmoc-Ala-2-CTC resin was measured to be 0.66 mmol / g, with a weight of 75.77 g.
[0030] (2) Preparation of Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-OH
[0031] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of the Fmoc-Ala-2-CTC resin obtained in step (1), add it to the solid-phase reactor, wash with DMF for swelling, deprotect twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, wash with DMF 6 times, and the resin is positive by K test. Weigh 28.27 g (80 mmol) of Fmoc-Ile-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF, add DIC (15.15 g, 120 mmol) under ice bath, and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash with DMF 4 times. The same steps were followed to continue coupling Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, and Fmoc-Phe-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 51.34 g of Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-OH.
[0032] Example 2: Preparation of Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH
[0033] (1) Preparation of Fmoc-Ile-2-CTC resin
[0034] 181.82 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 200 mmol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and washed three times. Fmoc-Ile-OH (141.36 g, 400 mmol), DIEA (103.39 g, 800 mmol), and 3.0 L of DMF were added, and the reaction was carried out for 6 h. The mixture was dried, and methanol (19.2 g, 600 mmol), DIEA (25.85 g, 200 mmol), and 3.0 L of DMF were added. The mixture was reacted for 1 h, dried, and washed three times with DMF and DCM respectively. The Fmoc-Ile-2-CTC resin was then removed, dried, and the substitution degree of the Fmoc-Ile-2-CTC resin was determined to be 0.67 mmol / g, with a weight of 151.33 g.
[0035] (2) Preparation of Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH
[0036] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of the Fmoc-Ile-2-CTC resin obtained in step (1), add it to the solid-phase reactor, wash with DMF for swelling, deprotect twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, wash with DMF 6 times, and the resin is positive by K test. Weigh 28.27 g (80 mmol) of Fmoc-Leu-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF, add DIC (15.15 g, 120 mmol) under ice bath, and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash with DMF 4 times. The same steps were followed to continue coupling Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, and Fmoc-Ala-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 51.29 g of Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH.
[0037] Example 3: Preparation of Fmoc-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH
[0038] (1) Preparation of Fmoc-Leu-2-CTC resin
[0039] 272.73 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 300 mmol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and washed three times. Fmoc-Leu-OH (141.36 g, 400 mmol), DIEA (103.39 g, 800 mmol), and 3.0 L of DMF were added, and the reaction was carried out for 6 h. The mixture was dried under vacuum, and methanol (19.2 g, 600 mmol), DIEA (38.8 g, 300 mmol), and 3.0 L of DMF were added. The reaction was carried out for 1 h, dried under vacuum, and washed three times each with DMF and DCM. The Fmoc-Leu-2-CTC resin was removed, dried, and the substitution degree of the Fmoc-Leu-2-CTC resin was measured to be 0.67 mmol / g, with a weight of 151.33 g.
[0040] (2) Preparation of Fmoc-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH
[0041] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of the Fmoc-Leu-2-CTC resin obtained in step (1), add it to the solid-phase reactor, wash with DMF for swelling, deprotect twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, wash with DMF 6 times, and the resin is positive by K test. Weigh 42.13 g (80 mmol) of Fmoc-Trp(Boc)-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF, add DIC (15.15 g, 120 mmol) under ice bath, and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash with DMF 4 times. The same steps were followed to continue coupling Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, and Fmoc-Lys(Alloc)-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 56.52 g of Fmoc-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH.
[0042] Example 4: Preparation of Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH
[0043] (1) Preparation of Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH
[0044] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of Fmoc-Ile-2-CTC resin obtained in step (1) of Example 2, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, wash with DMF 6 times, and the resin is positive by K test. Weigh 28.27 g (80 mmol) of Fmoc-Leu-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF, add DIC (15.15 g, 120 mmol) under ice bath, and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash with DMF 4 times. The same steps were followed to continue coupling Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, and Fmoc-Phe-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 49.89 g of Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH.
[0045] Example 5: Preparation of Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH
[0046] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of Fmoc-Leu-2-CTC resin from step (1) of Example 3, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, wash with DMF 6 times, and the resin is positive by K test. Weigh 42.13 g (80 mmol) of Fmoc-Trp(Boc)-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF, add DIC (15.15 g, 120 mmol) under ice bath, and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash with DMF 4 times. The same steps were followed to continue coupling Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, and Fmoc-Ala-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 49.33 g of Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH.
[0047] Example 6: Preparation of Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH
[0048] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of Fmoc-Leu-2-CTC resin from step (1) of Example 3, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, wash with DMF 6 times, and the resin is positive by K test. Weigh 42.13 g (80 mmol) of Fmoc-Trp(Boc)-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF, add DIC (15.15 g, 120 mmol) under ice bath, and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash with DMF 4 times. The same steps were followed to continue coupling Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, and Fmoc-Phe-OH. After the reaction was complete, the resin was washed three times with DMF and DCM respectively, and dried to obtain Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 46.73 g of Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH.
[0049] Example 7 Preparation of Fmoc-Thr(tBu)-Phe-OH
[0050] (1) Preparation of Fmoc-Phe-2-CTC resin
[0051] 90.91 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 100 mmol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and washed three times. Fmoc-Phe-OH (77.49 g, 200 mmol), DIEA (51.70 g, 400 mmol), and 1.5 L of DMF were added, and the reaction was carried out for 6 h. The mixture was dried under vacuum, and methanol (9.61 g, 300 mmol), DIEA (12.93 g, 100 mmol), and 1.5 L of DMF were added. The reaction was carried out for 1 h, dried under vacuum, and washed three times each with DMF and DCM. The Fmoc-Phe-2-CTC resin was removed, dried, and the substitution degree of the Fmoc-Phe-2-CTC resin was measured to be 0.67 mmol / g, with a weight of 77.63 g.
[0052] (2) Preparation of Fmoc-Thr(tBu)-Phe-OH
[0053] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of Fmoc-Phe-2-CTC resin obtained in step (1), add it to a solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, wash 6 times with DMF, and the resin is positive by K test. Weigh 31.80 g (80 mmol) of Fmoc-Thr(tBu)-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF, add DIC (15.15 g, 120 mmol) under ice bath, and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution, wash 4 times with DMF and 3 times with DCM, and dry to obtain Fmoc-Thr(tBu)-Phe-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness, and then dried under vacuum to obtain 19.84 g of Fmoc-Thr(tBu)-Phe-OH.
[0054] Example 8 Preparation of Fmoc-Leu-Asp(OtBu)-OH
[0055] (1) Preparation of Fmoc-Asp(OtBu)-2-CTC resin
[0056] 90.91 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 100 mmol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and washed three times. Fmoc-Asp(OtBu)-OH (82.29 g, 200 mmol), DIEA (51.70 g, 400 mmol), and 1.5 L of DMF were added, and the reaction was carried out for 6 h. The mixture was dried under vacuum, and methanol (9.61 g, 300 mmol), DIEA (12.93 g, 100 mmol), and 1.5 L of DMF were added. The reaction was carried out for 1 h, dried under vacuum, and washed three times each with DMF and DCM. The Fmoc-Asp(OtBu)-2-CTC resin was removed, dried, and the substitution degree of the Fmoc-Asp(OtBu)-2-CTC resin was measured to be 0.65 mmol / g, with a weight of 79.41 g.
[0057] (2) Preparation of Fmoc-Leu-Asp(OtBu)-OH
[0058] Weigh 61.54 g (0.65 mmol / g, 40 mmol) of Fmoc-Asp(OtBu)-2-CTC resin obtained in step (1), add it to a solid-phase reactor, wash with DMF for swelling, deprotect twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, wash with DMF 6 times, and the resin is positive by K test. Weigh 28.27 g (80 mmol) of Fmoc-Leu-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF, add DIC (15.15 g, 120 mmol) under ice bath, and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution, wash with DMF 4 times, wash with DCM 3 times, and dry to obtain Fmoc-Leu-Asp(OtBu)-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness, and then dried under vacuum to obtain 19.84 g of Fmoc-Leu-Asp(OtBu)-OH.
[0059] Example 9 Preparation of Fmoc-Gly-Gly-OH
[0060] (1) Preparation of Fmoc-Gly-2-CTC resin
[0061] 90.91 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 100 mmol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and washed three times. Fmoc-Gly-OH (59.46 g, 200 mmol), DIEA (51.70 g, 400 mmol), and 1.5 L of DMF were added, and the reaction was carried out for 6 h. The mixture was dried under vacuum, and methanol (9.61 g, 300 mmol), DIEA (12.93 g, 100 mmol), and 1.5 L of DMF were added. The reaction was carried out for 1 h, dried under vacuum, and washed three times each with DMF and DCM. The Fmoc-Gly-2-CTC resin was removed, dried, and the substitution degree of the Fmoc-Gly-2-CTC resin was measured to be 0.66 mmol / g, with a weight of 77.45 g.
[0062] (2) Preparation of Fmoc-Gly-Gly-OH
[0063] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Gly-2-CTC resin obtained in step (1), add it to a solid-phase reactor, wash with DMF for swelling, deprotect twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, wash with DMF 6 times, and the resin is positive by K test. Weigh 23.78 g (80 mmol) of Fmoc-Gly-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution, wash with DMF 4 times and DCM 3 times, and dry to obtain Fmoc-Gly-Gly-2-CTC resin. Add the resin to 1 L of 20% TFE / DCM (v / v) and react for 2 h. Filter, evaporate the filtrate to dryness, and vacuum dry to obtain 12.97 g of Fmoc-Gly-Gly-OH.
[0064] Example 10 Preparation of Fmoc-Ser(tBu)-Ser(tBu)-OH
[0065] (1) Preparation of Fmoc-Ser(tBu)-2-CTC resin
[0066] 90.91 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 100 mmol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (76.69 g, 200 mmol), DIEA (51.70 g, 400 mmol), and 1.5 L of DMF were added, and the reaction was carried out for 6 h. The mixture was dried under vacuum, and methanol (9.61 g, 300 mmol), DIEA (12.93 g, 100 mmol), and 1.5 L of DMF were added. The reaction was carried out for 1 h, dried under vacuum, and washed three times each with DMF and DCM. The Fmoc-Ser(tBu)-2-CTC resin was removed, dried, and the substitution degree of the Fmoc-Ser(tBu)-2-CTC resin was measured to be 0.66 mmol / g, with a weight of 76.45 g.
[0067] (2) Preparation of Fmoc-Ser(tBu)-Ser(tBu)-OH
[0068] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Ser(tBu)-2-CTC resin obtained in step (1), add it to a solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, wash 6 times with DMF, and the resin is positive by K test. Weigh 30.68 g (80 mmol) of Fmoc-Ser(tBu)-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF, add DIC (15.15 g, 120 mmol) under ice bath, and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution, wash 4 times with DMF and 3 times with DCM, and dry to obtain Fmoc-Ser(tBu)-Ser(tBu)-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness, and then dried under vacuum to obtain 19.01 g of Fmoc-Ser(tBu)-Ser(tBu)-OH.
[0069] Example 11 Preparation of Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH
[0070] (1) Preparation of Fmoc-AEEA-2-CTC resin
[0071] 90.91 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 100 mmol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and washed three times. Fmoc-AEEA-OH (77.08 g, 200 mmol), DIEA (51.70 g, 400 mmol), and 540 mL of DMF were added, and the reaction was carried out for 6 h. The mixture was dried under vacuum, and methanol (48.06 g, 1500 mmol), DIEA (12.92 g, 100 mmol), and 540 mL of DMF were added. The reaction was carried out for 1 h, dried under vacuum, and washed three times each with DMF and DCM. The Fmoc-AEEA-2-CTC resin was then removed, dried, and its substitution degree was measured to be 0.69 mmol / g, with a mass of 78.98 g.
[0072] (2) Preparation of AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)
[0073] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of Fmoc-AEEA-2-CTC resin from step (1) and add it to a solid-phase reactor. Swell and wash with DMF, then deprotect twice with 20% piperidine / DMF solution (v / v), first for 5 min and second for 15 min, followed by 6 washes with DMF. Weigh 30.83 g (80 mmol) of Fmoc-AEEA-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 360 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath conditions and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. Dry the mixture under vacuum and wash with DMF 6 times. The same steps were followed to continue coupling Fmoc-γGlu(α-OtBu)-OH and eicosanedioic acid monotert-butyl ester. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Eicosaned(mon-tBu)-γGlu(α-OtBu)-AEEA-AEEA-2-CTC resin. The resin was added to 1500 mL of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 32.48 g of AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu).
[0074] (3) Preparation of Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH
[0075] Weigh 32.48 g (40 mmol) of AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu) and 7.74 g (42 mmol) of PFP-OH obtained in step (2), add 100 mL of dichloromethane, cool to 0 °C, and add EDCI (11.5 g, 60 mmol) in six batches with stirring. Continue the reaction for 15 min, then raise the temperature to 25 °C and react for 2 h. Monitor the reaction of the starting materials by TLC and HPLC to ensure complete reaction. Stop the reaction, wash the reaction solution with 100 mL of water, 100 mL of saturated sodium bicarbonate solution, and 100 mL of saturated brine, respectively. Dry the organic phase with anhydrous sodium sulfate, filter, and evaporate to dryness to obtain an oily viscous substance. Weigh 14.73 g (40 mmol) of Fmoc-Lys-OH and dissolve it in 100 mL of 10% sodium carbonate aqueous solution. Add 100 mL of tetrahydrofuran and, with stirring, slowly add 200 mL of the above oily viscous substance to the tetrahydrofuran solution using a constant pressure dropping funnel at 5 °C. After the addition is complete, raise the temperature to 25 °C and continue the reaction for 3 h. Monitor the reaction of the starting material by TLC and HPLC to ensure complete reaction. Stop the reaction, adjust the pH to 3-4 with 1 M hydrochloric acid solution, evaporate the tetrahydrofuran to dryness, extract with DCM in aqueous phase, dry and concentrate the organic phase to obtain crude Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH. Purify to obtain 28 g of pure product, yield 59%, purity 99.2%.
[0076] Example 12 Preparation of crude Tirzepatide peptide 1
[0077] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, followed by six washes with DMF. The ninhydrin test result was positive.
[0078] Repeat the above steps, and sequentially couple the remaining amino acids or amino acid fragments according to the Tirzepatide peptide sequence, wherein the amino acid fragments are Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-OH prepared in Example 1 and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH prepared in Example 11, to obtain 39.42g of Tirzepatide peptide resin.
[0079] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 12.02 g of crude Tirzepatide peptide with a purity of 73.4%.
[0080] Example 13 Preparation of crude Tirzepatide peptide 2
[0081] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, followed by six washes with DMF. The ninhydrin test result was positive.
[0082] Repeat the above steps, and sequentially couple the remaining amino acids or amino acid fragments according to the Tirzepatide peptide sequence, wherein the amino acid fragments are Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH obtained in Example 2 and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH obtained in Example 11, to obtain 38.85g of Tirzepatide peptide resin.
[0083] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 12.03 g of crude Tirzepatide peptide with a purity of 74.3%.
[0084] Example 14 Preparation of crude Tirzepatide peptide 3
[0085] 12.5 g of Rink Amide MBHA resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, followed by six washes with DMF. The ninhydrin test result was positive.
[0086] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the Tirzepatide main chain peptide sequence, wherein the amino acid fragment is Fmoc-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH obtained in Example 3, and Pd(PPh3)4 / DMF after de-Alloc is coupled with AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu) obtained in step (2) of Example 11, to obtain 38.41g of Tirzepatide peptide resin.
[0087] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 11.92 g of crude Tirzepatide peptide with a purity of 73.8%.
[0088] Example 15 Preparation of crude Tirzepatide peptide 4
[0089] 12.5 g of Sieber resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, followed by six washes with DMF. The ninhydrin test result was positive.
[0090] Repeat the above steps, and sequentially couple the remaining amino acids or amino acid fragments according to the Tirzepatide peptide sequence, wherein the amino acid fragments are Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH obtained in Example 4 and Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH obtained in Example 11, to obtain 38.89g of Tirzepatide peptide resin.
[0091] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 11.97 g of crude Tirzepatide peptide with a purity of 73.6%.
[0092] Example 16 Preparation of crude Tirzepatide peptide 5
[0093] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, followed by six washes with DMF. The ninhydrin test result was positive.
[0094] Repeat the above steps, and sequentially couple the remaining amino acids or amino acid fragments according to the Tirzepatide peptide sequence, wherein the amino acid fragments are Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH obtained in Example 5 and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH obtained in Example 11, to obtain 38.93g of Tirzepatide peptide resin.
[0095] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 11.86 g of crude Tirzepatide peptide with a purity of 73.2%.
[0096] Example 17 Preparation of crude Tirzepatide peptide 6
[0097] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, followed by six washes with DMF. The ninhydrin test result was positive.
[0098] Repeat the above steps, and sequentially couple the remaining amino acids or amino acid fragments according to the Tirzepatide peptide sequence, wherein the amino acid fragments are Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH obtained in Example 6 and Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH obtained in Example 11, to obtain 38.46g of Tirzepatide peptide resin.
[0099] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 11.99 g of crude Tirzepatide peptide with a purity of 73.7%.
[0100] Example 18 Preparation of crude Tirzepatide peptide 7
[0101] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, followed by six washes with DMF. The ninhydrin test result was positive.
[0102] Repeat the above steps, and sequentially couple the remaining amino acids or amino acid fragments according to the Tirzepatide peptide sequence, wherein the amino acid fragments are Fmoc-Gly-Gly-OH obtained in Example 9, Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-OH obtained in Example 1, and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH obtained in Example 11, to obtain 38.76g of Tirzepatide peptide resin.
[0103] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 12.03 g of crude Tirzepatide peptide with a purity of 74.5%.
[0104] Example 19 Preparation of crude Tirzepatide peptide 8
[0105] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, followed by six washes with DMF. The ninhydrin test result was positive.
[0106] Repeat the above steps, and sequentially couple the remaining amino acids or amino acid fragments according to the Tirzepatide peptide sequence, wherein the amino acid fragments are Fmoc-Gly-Gly-OH prepared in Example 9, Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH prepared in Example 6, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioicacid(mon-tBu)]-OH prepared in Example 11, and Fmoc-Leu-Asp(OtBu)-OH prepared in Example 8, to obtain 38.99g of Tirzepatide peptide resin.
[0107] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 12.09 g of crude Tirzepatide peptide with a purity of 74.8%.
[0108] Example 20 Preparation of crude Tirzepatide peptide 9
[0109] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, followed by six washes with DMF. The ninhydrin test result was positive.
[0110] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the Tirzepatide main chain peptide sequence, wherein the amino acid fragments are Fmoc-Ser(tBu)-Ser(tBu)-OH prepared in Example 10, Fmoc-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH prepared in Example 3, and Fmoc-Thr(tBu)-Phe-OH prepared in Example 7. After de-Allocing Pd(PPh3)4 / DMF, couple the AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu) fragment prepared in step (2) of Example 11 to obtain 38.41g of Tirzepatide peptide resin.
[0111] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 11.94 g of crude Tirzepatide peptide with a purity of 74.0%.
[0112] Example 21 Preparation of crude Tirzepatide peptide 10
[0113] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v), the first time for 5 min and the second time for 15 min, followed by six washes with DMF. The ninhydrin test result was positive.
[0114] Repeat the above steps, and sequentially couple the remaining amino acids or amino acid fragments according to the Tirzepatide peptide sequence, wherein the amino acid fragments are Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH prepared in Example 4, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH prepared in Example 11, and Fmoc-Leu-Asp(OtBu)-OH prepared in Example 8, to obtain 38.71g of Tirzepatide peptide resin.
[0115] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether and dried. The resulting white solid was 12.01 g of crude Tirzepatide peptide with a purity of 73.9%.
[0116] Example 22 Preparation of Tirzepatide peptide 1
[0117] The crude Tirzepatide peptide obtained in Example 12 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 6.98 g of refined Tirzepatide peptide with an HPLC purity of 99.1% and a yield of 29.0%.
[0118] Example 23 Preparation of Tirzepatide peptide 2
[0119] The crude Tirzepatide peptide obtained in Example 13 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then linearly eluted by HPLC using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 7.51 g of refined Tirzepatide peptide with an HPLC purity of 99.5% and a yield of 31.2%.
[0120] Example 24 Preparation of Tirzepatide peptide 3
[0121] The crude Tirzepatide peptide obtained in Example 14 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then linearly eluted by HPLC using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 7.39 g of refined Tirzepatide peptide with an HPLC purity of 99.3% and a yield of 30.5%.
[0122] Example 25 Preparation of Tirzepatide peptide 4
[0123] The crude Tirzepatide peptide obtained in Example 15 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 7.59 g of refined Tirzepatide peptide with an HPLC purity of 99.2% and a yield of 31.3%.
[0124] Example 26 Preparation of Tirzepatide peptides 5
[0125] The crude Tirzepatide peptide obtained in Example 16 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 7.12 g of refined Tirzepatide peptide with an HPLC purity of 99.1% and a yield of 29.3%.
[0126] Example 27 Preparation of Tirzepatide peptides 6
[0127] The crude Tirzepatide peptide obtained in Example 17 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then linearly eluted by HPLC using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 7.82 g of refined Tirzepatide peptide with an HPLC purity of 99.4% and a yield of 32.3%.
[0128] Example 28 Preparation of Tirzepatide peptides 7
[0129] The crude Tirzepatide peptide obtained in Example 18 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 8.13 g of refined Tirzepatide peptide with an HPLC purity of 99.5% and a yield of 33.6%.
[0130] Example 29 Preparation of Tirzepatide peptides 8
[0131] The crude Tirzepatide peptide obtained in Example 19 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 8.18 g of refined Tirzepatide peptide with an HPLC purity of 99.4% and a yield of 33.8%.
[0132] Example 30 Preparation of Tirzepatide peptides 9
[0133] The crude Tirzepatide peptide obtained in Example 20 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 7.87 g of refined Tirzepatide peptide with an HPLC purity of 99.2% and a yield of 32.5%.
[0134] Example 31 Preparation of Tirzepatide peptides 10
[0135] The crude Tirzepatide peptide obtained in Example 21 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then linearly eluted by HPLC using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 7.94 g of refined Tirzepatide peptide with an HPLC purity of 99.1% and a yield of 32.8%.
Claims
1. A method for preparing Tirzepatide, characterized in that, Mainly includes: using Tirzepatide peptide resin was prepared by solid-phase peptide synthesis, and Tirzepatide was obtained by cleavage of the Tirzepatide peptide resin; the peptide fragments used were: Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH; or Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH; or Fmoc-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu–OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH; or Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH; or Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH; or Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH; or or Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH, or OR OR Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH, Fmoc-Leu-Asp(OtBu)-OH.
2. The method for preparing Tirzepatide according to claim 1, characterized in that, The peptide fragments used are: Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-OH and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH.
3. The method for preparing Tirzepatide according to claim 1, characterized in that, The peptide fragments used are: Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH.
4. The method for preparing Tirzepatide according to claim 1, characterized in that, The peptide fragments used are: Fmoc-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu–OH and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH.
5. The method for preparing Tirzepatide according to claim 1, characterized in that, The peptide fragments used are: Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-OH and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH.
6. The method for preparing Tirzepatide according to claim 1, characterized in that, The peptide fragments used are: Fmoc-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu–OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH, Fmoc-Ser(tBu)-Ser(tBu)-OH, and Fmoc-Thr(tBu)-Phe-OH.
7. The method for preparing Tirzepatide according to any one of claims 1-6, characterized in that, The prepared Tirzepatide was further purified and freeze-dried to obtain pure Tirzepatide.