A process for the preparation of tirzepatide

Abstract

The present application relates to the technical fields of polypeptide drug synthesis, and discloses a preparation method of Tirzepatide, which comprises the following steps: preparing Tirzepatide peptide resin by a solid-phase polypeptide synthesis method, and obtaining Tirzepatide by cleaving the Tirzepatide peptide resin; wherein the method for introducing Aib is: using 2-6 peptide fragments containing Aib. The present application can significantly reduce the generation of related impurities, guarantee the yield, and facilitate purification. In addition, the synthesis of multiple fragments can be carried out simultaneously, the synthesis period is shortened, and the present application is suitable for large-scale industrial production, and is a Tirzepatide preparation method with wide practical value and application prospect.

Description

Technical Field

[0001] This invention relates to the field of polypeptide drug synthesis technology, and in particular to a method for preparing Tirzepatide. Background Technology

[0002] Tirzepatide, developed by Eli Lilly, is a GIP-GLP1 dual receptor agonist that improves pancreatic β-cell function and insulin sensitivity in patients with type 2 diabetes. It has a positive effect on glycemic control and weight loss, and tolerability improves with increasing dose. It can also improve markers of non-alcoholic steatohepatitis (NASH) and reduce gastrointestinal side effects. Current data show that Tirzepatide is effective in lowering blood sugar and reducing weight, and it can also be used to treat other diseases.

[0003] Tirzepatide has the following structure:

[0004] 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-Eicosanedioic acid)-Ala-Phe-Val-Gln-Trp 25 -Leu-Ile-Ala-Gly-Gly 30 -Pro-Ser-Ser-Gly-Ala 35 -Pro-Pro-Pro-Ser-NH2.

[0005] Methods for preparing tirzepatide have been reported. Patent CN107207576A discloses a solid-phase preparation method for 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. Tirzepatide contains 39 amino acid residues and a relatively long side chain. The stepwise synthesis method has the problems of many steps, long cycle, many impurities, and difficult purification. Patent WO2020159949 discloses a solid-liquid phase combined method for preparing tirzepatide. The method involves first solid-phase synthesis of fragments of different lengths, then liquid-phase condensation of the fragments to obtain a fully protected peptide, and finally cleavage to obtain the polypeptide product. This method can reduce the risk of purification difficulties caused by the generation of related missing peptide impurities. However, the liquid-phase reaction is not easy to control, the intermediates are not easy to purify, and it is not conducive to large-scale industrial production.

[0006] Therefore, there is a need to find a method for preparing Tirzepatide that has a shorter synthesis cycle, fewer impurities, is easy to purify, and is suitable for large-scale production. Summary of the Invention

[0007] This invention addresses the problems of numerous steps, long cycles, numerous impurities, and difficult purification in existing technologies for the synthesis of Tirzepatide by providing a novel method for synthesizing Tirzepatide. The method includes the following steps: preparing Tirzepatide peptide resin using a solid-phase peptide synthesis method; cleaving the Tirzepatide peptide resin to obtain Tirzepatide; wherein the method for incorporating Aib is to use a 2-6 peptide fragment containing Aib.

[0008] Furthermore, in the process of synthesizing Tirzepatide using the above method, the Aib-containing 2-6 peptide fragments are selected from Tyr-Aib-Glu-Gly-Thr-Phe, Tyr-Aib-Glu-Gly-Thr, Tyr-Aib-Glu-Gly, Tyr-Aib-Glu, Tyr-Aib, Ile-Aib-Leu-Asp-Lys-Ile, Ser-Ile-Aib-Leu-Asp-Lys, Tyr-Ser-Ile-Aib-Leu-Asp, Asp-Ty r-Ser-Ile-Aib-Leu,Ser-Asp-Tyr-Ser-Ile-Aib,Ile-Aib-Leu-Asp-Lys,Ser-Ile-Aib-Leu-Asp,Tyr-Ser-Ile-Aib-Leu , Asp-Tyr-Ser-Ile-Aib, Ile-Aib-Leu-Asp, Ser-Ile-Aib-Leu, Tyr-Ser-Ile-Aib, Ile-Aib-Leu, Ser-Ile-Aib, Ile-Aib.

[0009] In a preferred embodiment of the present invention, the peptide fragment used for the second insertion of Aib is Tyr-Aib; the peptide fragment used for the thirteenth insertion of Aib is Ile-Aib.

[0010] In a preferred embodiment of the present invention, the peptide fragment used for the second insertion of Aib is Tyr-Aib-Glu-Gly; and the peptide fragment used for the thirteenth insertion of Aib is Ile-Aib.

[0011] In a preferred embodiment of the present invention, the peptide fragment used for the second insertion of Aib is: Tyr-Aib-Glu-Gly-Thr-Phe; the peptide fragment used for the thirteenth insertion of Aib is: Ile-Aib-Leu-Asp.

[0012] In a preferred embodiment of the present invention, the peptide fragment used for the second insertion of Aib is: Tyr-Aib-Glu-Gly-Thr-Phe; the peptide fragment used for the thirteenth insertion of Aib is: Ile-Aib.

[0013] In a preferred embodiment of the present invention, the peptide fragment used for the second insertion of Aib is Tyr-Aib-Glu-Gly; and the peptide fragment used for the thirteenth insertion of Aib is Ile-Aib-Leu-Asp.

[0014] In a preferred embodiment of the present invention, the peptide fragment used for the second insertion of Aib is: Tyr-Aib-Glu-Gly-Thr-Phe; the peptide fragment used for the thirteenth insertion of Aib is: Ile-Aib-Leu-Asp-Lys-Ile.

[0015] In a preferred embodiment of the present invention, during the synthesis of Tirzepatide, a dipeptide fragment or a combination thereof is used, wherein the dipeptide fragment is selected from Thr-Phe, Leu-Asp, Gly-Gly, and Ser-Ser.

[0016] Furthermore, the prepared Tirzepatide was further purified and freeze-dried to obtain pure Tirzepatide.

[0017] Due to steric hindrance, coupling Aib itself is difficult, and coupling with adjacent amino acids is also challenging, prolonging coupling time and introducing racemic impurities and deletion peptides. Furthermore, the structure of Aib renders its K and C detection methods unsuitable. Therefore, this invention uses 2-6 peptide fragments containing Aib for solid-phase coupling, facilitating in-process control and reducing difficulties in coupling with related amino acids (such as Tyr). 1 Aib 2 Glu 3 Gly 4 Ile 12 Aib 13 Leu 14 Deletion peptides or racemic impurities (such as Thr) introduced by factors such as long coupling time 5 Thr 7 This addresses issues such as yield and purification, while ensuring high yield. Furthermore, multiple fragments can be synthesized simultaneously, shortening the synthesis cycle and making it suitable for large-scale industrial production. Detailed Implementation

[0018] 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.

[0019] Example 1: Preparation of Boc-Tyr(tBu)-Aib-OH

[0020] (1) Preparation of Fmoc-Aib-2-CTC resin

[0021] 727.27 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 0.8 mol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Aib-OH (650 g, 2.0 mmol), DIEA (518 g, 4.0 mmol), and 5.4 L of DMF were added, and the reaction was carried out for 6 h. The mixture was dried under vacuum, and methanol (480 g, 15 mmol), DIEA (130 g, 1.0 mmol), and 5.4 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-Aib-2-CTC resin was removed, dried, and its substitution degree was measured to be 0.67 mmol / g, with a mass of 603.23 g.

[0022] (2) Synthesis of Boc-Tyr(tBu)-Aib-OH

[0023] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of the Fmoc-Aib-2-CTC resin obtained in step (1), add it to the solid-phase reactor, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), and wash 6 times with DMF. The ninhydrin test result was positive. Weigh Boc-Tyr(tBu)-OH (9.19 g, 20 mmol) and HOAt (3.24 g, 24 mmol) and dissolve them in 90 mL of DMF. Add DIC (3.78 g, 30 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The ninhydrin test result was negative. Dry the solution, wash 3 times with DMF and DCM respectively, and dry to obtain Boc-Tyr(tBu)-Aib-2-CTC resin. The resin was added to 300 mL 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 14.56 g of Boc-Tyr(tBu)-Aib-OH.

[0024] Example 2: Preparation of Fmoc-Ile-Aib-OH

[0025] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of Fmoc-Aib-2-CTC resin obtained in step (1) of Example 1, add it to a solid-phase reactor, wash with DMF for swelling, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), and wash 6 times with DMF. Weigh 28.27 g (80 mmol), HOBt (12.97 g, 96 mmol), and HBTU (45.48 g, 120 mmol) and dissolve them in 150 mL of DMF. Add DIEA (30.96 g, 240 mmol) under ice bath conditions. Add the solution to the solid-phase reactor and react for 2 h. Dry under vacuum, wash 3 times with DMF and DCM respectively, and dry to obtain Fmoc-Ile-Aib-2-CTC resin. Add the resin to 1000 mL of 20% TFE / DCM (v / v) and react for 2 h. Filter, evaporate the filtrate to dryness, and vacuum dry to obtain 14.27 g of Fmoc-Ile-Aib-OH.

[0026] Example 3: Preparation of Boc-Tyr(tBu)-Aib-Glu(OtBu)-OH

[0027] (1) Preparation of Fmoc-Glu(OtBu)-2-CTC resin

[0028] 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-Glu(OtBu)-OH (85.10 g, 200 mmol), DIEA (51.70 g, 400 mmol), and 360 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 360 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-Glu(OtBu)-2-CTC resin was then removed, dried, and its substitution degree was measured to be 0.62 mmol / g, with a mass of 77.42 g.

[0029] (2) Preparation of Boc-Tyr(tBu)-Aib-Glu(OtBu)-OH

[0030] Weigh 64.52 g (0.62 mmol / g, 40 mmol) of the Fmoc-Glu(OtBu)-2-CTC resin obtained in step (1), add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and the resin shows a positive result for K detection. Weigh 26.03 g (80 mmol), HOBt (12.97 g, 96 mmol) and HBTU (45.48 g, 120 mmol) and dissolve them in 150 mL of DMF, add DIEA (30.96 g, 240 mmol) under ice bath. Add the solution to the solid-phase reactor, react for 2 h, the resin shows a negative result for K detection, dry under vacuum, and wash with DMF 6 times. The same steps were followed to continue coupling Boc-Tyr(tBu)-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Boc-Tyr(tBu)-Aib-Glu(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 20.97 g of Boc-Tyr(tBu)-Aib-Glu(OtBu)-OH.

[0031] Example 4: Preparation of Fmoc-Ile-Aib-Leu-OH

[0032] (1) Preparation of Fmoc-Leu-2-CTC resin

[0033] 364 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 0.4 mol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Leu-OH (283 g, 0.8 mol), DIEA (207 g, 0.8 mol), and 2.1 L of DMF were added, and the reaction was carried out for 6 h. The mixture was dried, and methanol (192 g, 6.0 mol), DIEA (52 g, 0.4 mol), and 2.1 L of DMF were added. The reaction was carried out for 1 h, dried, and washed three times with DMF and DCM respectively. The Fmoc-Leu-2-CTC resin was removed, dried, and its substitution degree was measured to be 0.65 mmol / g, with a mass of 311 g.

[0034] (2) Synthesis of Fmoc-Ile-Aib-Leu-OH

[0035] Weigh 61.54 g (0.65 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) (5 min + 15 min), wash with DMF 6 times, and the resin is positive by K test. Weigh 26.03 g (80 mmol) of Fmoc-Aib-OH and HOAt (12.97 g, 96 mmol) 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 positive by K test. Dry the solution and wash with DMF 6 times.

[0036] The same steps were followed to continue coupling Fmoc-Ile-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Ile-Aib-Leu-2-CTC resin. The resin was added to 1000 mL 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 18.98 g of Fmoc-Ile-Aib-Leu-OH.

[0037] Example 5: Preparation of Fmoc-Ser(tBu)-Ile-Aib-OH

[0038] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of Fmoc-Aib-2-CTC resin obtained in step (1) of Example 1, add it to a solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), and wash 6 times with DMF. Weigh 28.27 g (80 mmol), HOBt (12.97 g, 96 mmol) and HBTU (45.48 g, 120 mmol) and dissolve them in 150 mL of DMF. Add DIEA (30.96 g, 240 mmol) under ice bath conditions. Add the solution to the solid-phase reactor, react for 2 h, dry under vacuum, and wash 6 times with DMF.

[0039] The same steps were followed to continue coupling Fmoc-Ser(tBu)-OH to obtain Fmoc-Ser(tBu)-Ile-Aib-2-CTC resin. The resin was added to 1500 mL of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness under vacuum to obtain 20.24 g of Fmoc-Ser(tBu)-Ile-Aib-OH.

[0040] Example 6: Preparation of Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH

[0041] (1) Preparation of Fmoc-Gly-2-CTC resin

[0042] 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 360 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 360 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-Gly-2-CTC resin was then removed, dried, and its substitution degree was measured to be 0.67 mmol / g, with a mass of 75.79 g.

[0043] (2) Preparation of Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH

[0044] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of the Fmoc-Gly-2-CTC resin obtained in step (1), add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and the resin is positive by K test. Weigh 34.04 g (80 mmol) of Fmoc-Glu(OtBu)-OH and HOAt (12.97 g, 96 mmol) 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 6 times. The same steps were followed to couple the Boc-Tyr(tBu)-Aib-OH prepared in Example 1. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-2-CTC resin. The resin was added to 1000 mL 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 23.91 g of Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH.

[0045] Example 7: Preparation of Fmoc-Ile-Aib-Leu-Asp(OtBu)-OH

[0046] (1) Preparation of Fmoc-Asp(OtBu)-2-CTC resin

[0047] 273 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 0.3 mol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and washed three times. Fmoc-Asp(OtBu)-OH (247 g, 0.6 mol), DIEA (155 g, 0.8 mol), and 1.6 L of DMF were added, and the reaction was carried out for 6 h. The mixture was then dried, and methanol (144 g, 4.5 mol), DIEA (39 g, 0.3 mol), and 1.6 L of DMF were added. The reaction was carried out for 1 h, dried, and washed three times with DMF and DCM respectively. The Fmoc-Asp(OtBu)-2-CTC resin was removed, dried, and its substitution degree was measured to be 0.65 mmol / g, with a mass of 234 g.

[0048] (2) Synthesis of Fmoc-Ile-Aib-Leu-Asp(OtBu)-OH

[0049] Weigh 61.54 g (0.65 mmol / g, 40 mmol) of the Fmoc-Asp(OtBu)-2-CTC resin obtained in step (1), add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 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 HOAt (12.97 g (96 mmol)) 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 6 times.

[0050] The same steps were followed to couple the Fmoc-Ile-Aib-OH prepared in Example 2. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Ile-Aib-Leu-Asp(OtBu)-2-CTC resin. The resin was added to 1000 mL 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 25.19 g of Fmoc-Ile-Aib-Leu-Asp(OtBu)-OH.

[0051] Example 8: Preparation of Fmoc-Ser(tBu)-Ile-Aib-Leu-OH

[0052] Weigh 61.54 g (0.65 mmol / g, 40 mmol) of Fmoc-Leu-2-CTC resin obtained in step (1) of Example 4, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and the resin is positive by K test. Weigh Fmoc-Aib-OH (26.03 g, 80 mmol), HOBt (12.97 g, 96 mmol) and HBTU (45.48 g, 120 mmol) and dissolve them in 150 mL of DMF, add DIEA (30.96 g, 240 mmol) under ice bath. Add the solution to the solid-phase reactor, react for 2 h, the resin is negative by K test, dry under vacuum, and wash with DMF 6 times. The same steps were followed to continue coupling Fmoc-Ile-OH and Fmoc-Ser(tBu)-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Ser(tBu)-Ile-Aib-Leu-2-CTC resin. The resin was added to 1000 mL 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 23.35 g of Fmoc-Ser(tBu)-Ile-Aib-Leu-OH.

[0053] Example 9: Preparation of Fmoc-Tyr(tBu)-Ser(tBu)-Ile-Aib-OH

[0054] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of Fmoc-Aib-2-CTC resin obtained in step (1) of Example 1, add it to a solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), and wash 6 times with DMF. Weigh 28.27 g (80 mmol) of Fmoc-Ile-OH and HOAt (12.97 g, 96 mmol) 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. Dry under vacuum and wash 6 times with DMF. The same steps were followed to continue coupling Fmoc-Ser(tBu)-OH and Fmoc-Tyr(tBu)-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Tyr(tBu)-Ser(tBu)-Ile-Aib-2-CTC resin. The resin was added to 1500 mL 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 27.87 g of Fmoc-Tyr(tBu)-Ser(tBu)-Ile-Aib-OH.

[0055] Example 10: Preparation of Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH

[0056] (1) Preparation of Fmoc-Thr(tBu)-2-CTC resin

[0057] 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-Thr(tBu)-OH (79.49 g, 200 mmol), DIEA (51.70 g, 400 mmol), and 360 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 360 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-Glu(OtBu)-2-CTC resin was then removed, dried, and its substitution degree was measured to be 0.67 mmol / g, with a mass of 75.79 g.

[0058] (2) Preparation of Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH

[0059] Weigh 58.82 g (0.68 mmol / g, 40 mmol) of the Fmoc-Thr(tBu)-2-CTC resin obtained in step (1), add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 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 and wash with DMF 6 times.

[0060] The same steps were followed to continue coupling Fmoc-Glu(OtBu)-OH and Boc-Tyr(tBu)-Aib-OH prepared in Example 1. After the reaction was complete, the resin was washed three times with DMF and DCM respectively, and dried to obtain Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-2-CTC resin. The resin was added to 1000 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 27.95 g of Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH.

[0061] Example 11: Preparation of Fmoc-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-OH

[0062] (1) Preparation of Fmoc-Lys(Boc)-2-CTC resin

[0063] 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-Lys(Boc)-OH (187.42 g, 400 mmol), DIEA (103.4 g, 800 mmol), and 720 mL of DMF were added, and the reaction was carried out for 6 h. The mixture was then dried under vacuum, and methanol (96.12 g, 3000 mmol), DIEA (25.84 g, 200 mmol), and 720 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-Lys(Boc)-2-CTC resin was removed, dried, and its substitution degree was measured to be 0.69 mmol / g, with a mass of 149.54 g.

[0064] (2) Preparation of Fmoc-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-OH

[0065] Weigh 57.97 g (0.69 mmol / g, 40 mmol) of the Fmoc-Lys(Boc)-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) (5 min + 15 min), wash with DMF 6 times, and test the resin for positive K. Continue coupling Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, and Fmoc-Ile-Aib-OH prepared in Example 2 using the same steps. After the reaction is complete, wash three times each with DMF and DCM, and dry to obtain Fmoc-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-2-CTC resin. Add the resin to 1500 mL of 20% TFE / DCM (v / v) and react for 2 h. Filter, evaporate the filtrate to dryness, and vacuum dry to obtain 31.97g of Fmoc-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-OH.

[0066] Example 12: Preparation of Fmoc-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-OH

[0067] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Asp(OtBu)-2-CTC resin obtained in step (1) of Example 7, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 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 6 times. The same steps were followed to couple Fmoc-Ile-Aib-OH and Fmoc-Ser(tBu)-OH prepared in Example 2. After the reaction was complete, the resin was washed three times with DMF and DCM respectively, and dried to obtain Fmoc-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-2-CTC resin. The resin was added to 1000 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 29.10 g of Fmoc-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-OH.

[0068] Example 13: Preparation of Fmoc-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-OH

[0069] Weigh 61.54 g (0.65 mmol / g, 40 mmol) of Fmoc-Leu-2-CTC resin obtained in step (1) of Example 4, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and the resin is positive by K test. Weigh Fmoc-Aib-OH (26.03 g, 80 mmol), HOBt (12.97 g, 96 mmol) and HBTU (45.48 g, 120 mmol) and dissolve them in 150 mL of DMF, add DIEA (30.96 g, 240 mmol) under ice bath. Add the solution to the solid-phase reactor, react for 2 h, the resin is negative by K test, dry under vacuum, and wash with DMF 6 times. The same steps were followed to continue coupling Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Tyr(tBu)-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-2-CTC resin. The resin was added to 1000 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 31.81 g of Fmoc-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-OH.

[0070] Example 14: Preparation of Fmoc-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-OH

[0071] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of Fmoc-Aib-2-CTC resin obtained in step (1) of Example 1, add it to a solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), and wash 6 times with DMF. Weigh 28.27 g (80 mmol), HOBt (12.97 g, 96 mmol) and HBTU (45.48 g, 120 mmol) and dissolve them in 150 mL of DMF. Add DIEA (30.96 g, 240 mmol) under ice bath conditions. Add the solution to the solid-phase reactor, react for 2 h, dry under vacuum, and wash 6 times with DMF. The same steps were followed to continue coupling Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-2-CTC resin. The resin was added to 1500 mL 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 32.74 g of Fmoc-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-OH.

[0072] Example 15: Preparation of Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-OH

[0073] (1) Preparation of Fmoc-Phe-2-CTC resin

[0074] 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 360 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 360 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-Phe-2-CTC resin was then removed, dried, and its substitution degree was measured to be 0.68 mmol / g, with a mass of 76.48 g.

[0075] (2) Preparation of Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-OH

[0076] Weigh 58.82 g (0.68 mmol / g, 40 mmol) of the Fmoc-Phe-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) (5 min + 15 min), wash with DMF 6 times, 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, 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, react for 2 h, the resin is negative by K test, dry under vacuum, and wash with DMF 6 times. The same steps were followed to continue coupling Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, and Boc-Tyr(tBu)-Aib-OH prepared in Example 1. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-2-CTC resin. The resin was added to 1000 mL of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness, and then vacuum dried to obtain 33.34 g of Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-OH.

[0077] Example 16: Preparation of Fmoc-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-OH

[0078] (1) Preparation of Fmoc-Ile-2-CTC resin

[0079] 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-Ile-OH (70.68 g, 200 mmol), DIEA (51.70 g, 400 mmol), and 360 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 360 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-Ile-2-CTC resin was then removed, dried, and its substitution degree was measured to be 0.66 mmol / g, with a mass of 78.67 g.

[0080] (2) Preparation of Fmoc-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-OH

[0081] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of the Fmoc-Ile-2-CTC resin obtained in step (1), add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and the resin is positive by K test. Weigh 37.48 g (80 mmol) of Fmoc-Lys(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 6 times. The same steps were followed to continue coupling Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, and Fmoc-Ile-Aib-OH prepared in Example 2. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-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 37.09 g of Fmoc-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-OH.

[0082] Example 17: Preparation of Fmoc-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-OH

[0083] Weigh 57.97 g (0.69 mmol / g, 40 mmol) of Fmoc-Lys(Boc)-2-CTC resin obtained in step (1) of Example 11, add it to a solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and the resin is positive by K test. Weigh 32.92 g (80 mmol) of Fmoc-Asp(OtBu)-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 6 times. The same steps were followed to continue coupling Fmoc-Leu-OH. The Fmoc-Ile-Aib-OH and Fmoc-Ser(tBu)-OH prepared in Example 2 were washed three times each with DMF and DCM after the reaction was complete, and dried to obtain Fmoc-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-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 vacuum dried to obtain 37.65 g of Fmoc-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-OH.

[0084] Example 18: Preparation of Fmoc-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-OH

[0085] Weigh 58.82 g (0.68 mmol / g, 40 mmol) of Fmoc-Asp(OtBu)-2-CTC resin obtained in step (1) of Example 7, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 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 6 times. The same steps were followed to couple Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Tyr(tBu)-OH obtained in Example 2. After the reaction was complete, the resin was washed three times with DMF and DCM, and dried to obtain Fmoc-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-2-CTC resin. The resin was added to 1500 mL of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness, and then vacuum dried to obtain Fmoc-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-OH 36.45.

[0086] Example 19: Preparation of Fmoc-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-OH

[0087] Weigh 61.54 g (0.65 mmol / g, 40 mmol) of Fmoc-Leu-2-CTC resin obtained in step (1) of Example 4, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and the resin is positive by K test. Weigh 26.03 g (80 mmol) of Fmoc-Aib-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 6 times. The same steps were followed to continue coupling Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH. After the reaction was complete, the resin was washed three times with DMF and DCM respectively, and dried to obtain Fmoc-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-2-CTC resin. The resin was added to 1500 mL 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 37.34 g of Fmoc-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-OH.

[0088] Example 20: Preparation of Fmoc-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-OH

[0089] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of Fmoc-Aib-2-CTC resin obtained in step (1) of Example 1, add it to a solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), and wash 6 times with DMF. Weigh 28.27 g (80 mmol) of Fmoc-Ile-OH and 12.97 g (96 mmol) of HOBt, 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, react for 2 h, dry under vacuum, and wash 6 times with DMF. The same steps were followed to continue coupling Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Ser(tBu)-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-2-CTC resin. The resin was added to 1500 mL 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 37.48 g of Fmoc-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-OH.

[0090] Example 21: Preparation of Fmoc-Thr(tBu)-Phe-OH

[0091] Weigh 58.82 g (0.68 mmol / g, 40 mmol) of Fmoc-Phe-2-CTC resin obtained in step (1) of Example 15, add it to a solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and K test shows the resin is positive. 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. K test shows the resin is negative. Dry the solution, wash 3 times with DMF and DCM respectively, and dry to obtain Fmoc-Thr(tBu)-Phe-2-CTC resin. The resin was added to 1000 mL 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 18.97 g of Fmoc-Thr(tBu)-Phe-OH.

[0092] Example 22: Preparation of Fmoc-Leu-Asp(OtBu)-OH

[0093] 61.54 g (0.65 mmol / g, 40 mmol) of Fmoc-Asp(OtBu)-2-CTC resin obtained in step (1) of Example 7 was weighed and added to a solid-phase reactor. The resin was swollen and washed with DMF, deprotected twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), washed 6 times with DMF, and tested positive for K. Fmoc-Leu-OH (28.27 g, 80 mmol) and HOBt (12.97 g, 96 mmol) were weighed and dissolved in 150 mL of DMF. DIC (15.15 g, 120 mmol) was added under ice bath conditions and activated for 3 min. The activated solution was added to the solid-phase reactor and reacted for 2 h. The resin tested negative for K. The solution was dried, washed 3 times each with DMF and DCM, and dried to obtain Fmoc-Leu-Asp(OtBu)-2-CTC resin. The resin was added to 1000 mL 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 18.61 g of Fmoc-Leu-Asp(OtBu)-OH.

[0094] Example 23: Preparation of Fmoc-Gly-Gly-OH

[0095] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of Fmoc-Gly-2-CTC resin obtained in step (1) of Example 6, add it to a solid-phase reactor, swell and wash with DMF, deprotect twice with 208% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and K test shows the resin is positive. Weigh 34.04 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. K test shows the resin is negative. Dry the solution, wash 3 times with DMF and DCM respectively, and dry to obtain Fmoc-Gly-Gly-2-CTC resin. The resin was added to 1000 mL 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 12.64 g of Fmoc-Gly-Gly-OH.

[0096] Example 24: Preparation of Fmoc-Ser(tBu)-Ser(tBu)-OH

[0097] (1) Preparation of Fmoc-Ser(tBu)-2-CTC resin

[0098] 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 360 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 360 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-Ser(tBu)-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.

[0099] (2) Preparation of Fmoc-Ser(tBu)-Ser(tBu)-OH

[0100] Weigh 57.97 g (0.69 mmol / g, 40 mmol) of the Fmoc-Ser(tBu)-2-CTC resin obtained in step (1), add it to a solid-phase reactor, wash with DMF for swelling, deprotect twice with 208% piperidine / DMF solution (v / v) (5 min + 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 3 times with DMF and DCM respectively, and dry to obtain Fmoc-Ser(tBu)-Ser(tBu)-2-CTC resin. The resin was added to 1000 mL 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 18.39 g of Fmoc-Ser(tBu)-Ser(tBu)-OH.

[0101] Example 25: Preparation of Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH

[0102] (1) Preparation of Fmoc-AEEA-2-CTC resin

[0103] 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.

[0104] (2) Preparation of AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)

[0105] Weigh 59.70 g (0.67 mmol / g, 40 mmol) of Fmoc-AEEA-2-CTC resin from step (1), add it to a solid-phase reactor, wash with DMF to swell, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), and wash 6 times with DMF. Weigh 30.83 g (80 mmol) of Fmoc-AEEA-OH and 12.97 g (96 mmol) of HOBt, dissolve them in 360 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, react for 2 h, dry under vacuum, and wash 6 times with DMF. 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).

[0106] (3) Preparation of Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH

[0107] 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%.

[0108] Example 26: Preparation of Tirzepatide 1

[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), HOBt (1.62 g, 12 mmol), and HBTU (5.70 g, 15 mmol) were weighed and dissolved in 60 mL of DMF. DIEA (3.87 g, 30 mmol) was added under ice bath conditions. The solution was added to the solid-phase reactor and reacted 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) (5 min + 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 peptide sequence, wherein the amino acid fragments are Fmoc-Lys[AEEA-AEEA-Glu(OtBu)-Eico(OtBu)]-OH prepared in Example 25, Fmoc-Ile-Aib-OH prepared in Example 2, and Boc-Tyr(tBu)-Aib-OH prepared in Example 1, to obtain 38.07g of Tirzepatide peptide resin.

[0111] The obtained Tirzepatide peptide resin was added to 380 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% and a yield of 58.3%.

[0112] The obtained crude Tirzepatide peptide 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 purified Tirzepatide was then freeze-dried to obtain 6.93 g of refined Tirzepatide peptide with an HPLC purity of 99.6% and a yield of 28.8%.

[0113] Example 27: Preparation of Tirzepatide 2

[0114] 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), HOBt (1.62 g, 12 mmol), and HBTU (5.70 g, 15 mmol) were weighed and dissolved in 60 mL of DMF. DIEA (3.87 g, 30 mmol) was added under ice bath conditions. The solution was added to the solid-phase reactor and reacted 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) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0115] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the peptide sequence, wherein the amino acid fragments are Fmoc-Lys[AEEA-AEEA-Glu(OtBu)-Eico(OtBu)]-OH prepared in Example 25, Fmoc-Ile-Aib-OH prepared in Example 2, and Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH prepared in Example 6, to obtain 38.68g of Tirzepatide peptide resin.

[0116] 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 74.5% and a yield of 57.9%.

[0117] The obtained crude Tirzepatide peptide 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 purified Tirzepatide was then freeze-dried to obtain 6.97 g of refined Tirzepatide peptide with an HPLC purity of 99.7% and an overall yield of 28.9%.

[0118] Example 28: Preparation of Tirzepatide 3

[0119] 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) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0120] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the peptide sequence, wherein the amino acid fragments are Fmoc-Lys[AEEA-AEEA-Glu(OtBu)-Eico(OtBu)]-OH prepared in Example 25, Fmoc-Ile-Aib-Leu-Asp(OtBu)-OH prepared in Example 7, and Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH prepared in Example 6, to obtain 37.94g of Tirzepatide peptide resin.

[0121] The obtained Tirzepatide peptide resin was added to 380 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 75.3% and a yield of 58.9%.

[0122] The obtained crude Tirzepatide peptide 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 purified Tirzepatide was then freeze-dried to obtain 6.89 g of refined Tirzepatide peptide with an HPLC purity of 99.6% and a yield of 28.6%.

[0123] Example 29: Preparation of Tirzepatide 4

[0124] 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), HOBt (1.62 g, 12 mmol), and HBTU (5.70 g, 15 mmol) were weighed and dissolved in 60 mL of DMF. DIEA (3.87 g, 30 mmol) was added under ice bath conditions. The solution was added to the solid-phase reactor and reacted 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) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0125] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the peptide sequence, wherein the amino acid fragments are Fmoc-Lys[AEEA-AEEA-Glu(OtBu)-Eico(OtBu)]-OH prepared in Example 25, Fmoc-Ile-Aib-OH prepared in Example 2, and Boc-Tyr(tBu)-Aib-Glu(Otbu)-Gly-Thr(tBu)-Phe-OH prepared in Example 15, to obtain 37.65g of Tirzepatide peptide resin.

[0126] The obtained Tirzepatide peptide resin was added to 380 mL of a 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 2000 mL 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 11.83 g of crude Tirzepatide peptide with a purity of 74.2% and a yield of 57.3%.

[0127] The obtained crude Tirzepatide peptide 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 purified Tirzepatide was then freeze-dried to obtain 6.99 g of refined Tirzepatide peptide with an HPLC purity of 99.7% and a yield of 29.1%.

[0128] Example 30: Preparation of Tirzepatide 5

[0129] 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) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0130] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the peptide sequence. The amino acid fragments are Fmoc-Lys[AEEA-AEEA-Glu(OtBu)-Eico(OtBu)]-OH prepared in Example 25, Fmoc-Ile-Aib-Leu-Asp(OtBu)-OH prepared in Example 7, and Boc-Tyr(tBu)-Aib-Glu(Otbu)-Gly-Thr(tBu)-Phe-OH prepared in Example 15, to obtain 38.41g of Tirzepatide peptide resin.

[0131] The obtained Tirzepatide peptide resin was added to 384.1 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 700 mL of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether. The resulting white solid was 12.05 g of crude Tirzepatide peptide with a purity of 73.8% and a yield of 58.0%.

[0132] The obtained crude Tirzepatide peptide 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 purified Tirzepatide was then freeze-dried to obtain 6.92 g of refined Tirzepatide peptide with an HPLC purity of 99.8% and a yield of 28.8%.

[0133] Example 31: Preparation of Tirzepatide 6

[0134] 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) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0135] Repeat the above steps, and sequentially couple the remaining amino acids or amino acid fragments according to the peptide sequence, wherein the amino acid fragments are Fmoc-Lys[AEEA-AEEA-Glu(OtBu)-Eico(OtBu)]-OH prepared in Example 25, Fmoc-Ile-Aib-Leu-Asp(Otbu)-Lys(Boc)-Ile-OH prepared in Example 16, and Boc-Tyr(tBu)-Aib-Glu(Otbu)-Gly-Thr(tBu)-Phe-OH prepared in Example 15, to obtain 37.95g of Tirzepatide peptide resin.

[0136] The obtained Tirzepatide peptide resin was added to 380 mL of a 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. The resulting white solid was 12.07 g of crude Tirzepatide peptide, with a purity of 74.1% and a yield of 58.3%.

[0137] The obtained crude Tirzepatide peptide 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 purified Tirzepatide was then freeze-dried to obtain 6.88 g of refined Tirzepatide peptide with an HPLC purity of 99.8% and a yield of 28.6%.

[0138] Example 32: Preparation of Tirzepatide 7

[0139] 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), HOBt (1.62 g, 12 mmol), and HBTU (5.70 g, 15 mmol) were weighed and dissolved in 60 mL of DMF. DIEA (3.87 g, 30 mmol) was added under ice bath conditions. The solution was added to the solid-phase reactor and reacted 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) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0140] Repeat the above steps, and sequentially couple the remaining amino acids or amino acid fragments according to the peptide sequence, wherein the amino acid fragments are Fmoc-Ser(tBu)-Ser(tBu)-OH prepared in Example 24, Fmoc-Gly-Gly-OH prepared in Example 23, Fmoc-Lys[AEEA-AEEA-Glu(OtBu)-Eico(OtBu)]-OH prepared in Example 25, Fmoc-Leu-Asp(OtBu)-OH prepared in Example 22, Fmoc-Ser(tBu)-Ile-Aib-OH prepared in Example 5, Fmoc-Thr(tBu)-Phe-OH prepared in Example 21, and Fmoc-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-OH prepared in Example 14, to obtain 38.05 g of Tirzepatide peptide resin.

[0141] The obtained Tirzepatide peptide resin was added to 180 mL of a 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 2000 mL of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether. The resulting white solid was 12.06 g of crude Tirzepatide peptide with a purity of 73.1% and a yield of 57.5%.

[0142] The obtained crude Tirzepatide peptide 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 purified Tirzepatide was then freeze-dried to obtain 6.95 g of refined Tirzepatide peptide with an HPLC purity of 99.7% and a yield of 28.9%.

[0143] Example 33: Preparation of Tirzepatide 8

[0144] 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) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0145] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the main chain peptide sequence. The amino acid fragments are Fmoc-Ile-Aib-Leu-OH prepared in Example 4 and Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH prepared in Example 10. The 20th amino acid residue and the protecting group are Lys(Alloc). After de-Allocing Pd(PPh3)4 / DMF, the fragment AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu) is coupled to obtain 38.34g of Tirzepatide peptide resin.

[0146] The obtained Tirzepatide peptide resin was added to 391.4 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 72.5% and a yield of 56.6%.

[0147] The obtained crude Tirzepatide peptide 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 purified Tirzepatide was then freeze-dried to obtain 6.70 g of refined Tirzepatide peptide with an HPLC purity of 99.1% and a yield of 27.9%.

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: Boc-Tyr(tBu)-Aib-OH, Fmoc-Ile-Aib-OH, Fmoc-Lys[AEEA-AEEA-Glu(OtBu)-Eico(OtBu)]-OH; or Fmoc-Ile-Aib-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH, Fmoc-Lys[AEEA-AEEA-Glu(OtBu)-Eico(OtBu)]-OH; or Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH、Fmoc-Ile-Aib-Leu-Asp(OtBu)-OH、 Fmoc-Lys[AEEA-AEEA-Glu(OtBu)-Eico(OtBu)]-OH; or or or or Fmoc-Ser(tBu)-Ile-Aib-OH, Fmoc-Asp (OtBu) -Tyr (tBu) -Ser (tBu) Or Fmoc-Ile-Aib-Leu-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH, wherein the 20th amino acid residue and protecting group are Lys(Alloc), and the Pd(PPh3)4 / DMF de-Alloc coupling fragment AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu).

2. The method for preparing Tirzepatide according to claim 1, characterized in that, The peptide fragments used are: Boc-Tyr(tBu)-Aib-OH, Fmoc-Ile-Aib-OH, Fmoc-Lys[AEEA-AEEA-Glu(OtBu)-Eico(OtBu)]-OH.

3. The method for preparing Tirzepatide according to claim 1, characterized in that, The peptide fragments used are: Fmoc-Ile-Aib-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH, Fmoc-Lys[AEEA-AEEA-Glu(OtBu)-Eico(OtBu)]-OH.

4. The method for preparing Tirzepatide according to claim 1, characterized in that, The peptide fragments used are: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH, Fmoc-Ile-Aib-Leu-Asp(OtBu)-OH, Fmoc-Lys[AEEA-AEEA-Glu(OtBu)-Eico(OtBu)]-OH.

5. The method for preparing Tirzepatide according to any one of claims 1 to 4, characterized in that, The prepared Tirzepatide was further purified and freeze-dried to obtain pure Tirzepatide.