A process for the preparation of tirzepatide

Abstract

The present application relates to the field of polypeptide medicine preparation, and discloses a preparation method of Tirzepatide, mainly comprising the following steps: (1) preparing Tirzepatide isopeptide resin by solid-phase polypeptide synthesis method, wherein, Thr or Ser in the Tirzepatide isopeptide resin is in the form of formula (1); (2) cleaving the Tirzepatide isopeptide resin obtained in step (1) to obtain Tirzepatide isopeptide; (3) stirring the Tirzepatide isopeptide obtained in step (2) in a neutral or alkaline aqueous solution to obtain Tirzepatide. The present application uses formula (1) to prepare Tirzepatide, which can reduce beta folding, reduce resin polycondensation, greatly improve the coupling effect of hydrophobic amino acids, finally reduce the generation of missing peptides, improve the yield and purity of the target product, and is a preparation method suitable for large-scale production of Tirzepatide.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide drug preparation, and in particular to a preparation method of Tirzepatide. Background Art

[0002] Diabetes is a chronic disease characterized by hyperglycemia caused by defects in insulin secretion, insulin action, or both. In type 2 diabetes, the combined effects of impaired insulin secretion and insulin resistance are associated with elevated blood sugar. US9474780B2 publicly reported Tirzepatide for the first time. Tirzepatide has dual receptor agonist effects on glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). It is a blockbuster product under development by Eli Lilly and Company and is currently undergoing phase III clinical trials. It is expected to be approved for marketing in 2022. It is considered to be the GLP-1 analog hypoglycemic drug most likely to pose a market challenge to semaglutide. 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 -Pro36 -Pro 37 -Pro 38 -Ser 39 -NH2.

[0003] CN107207576A discloses a solid phase method for preparing Tirzepatide, which involves the stepwise solid phase synthesis of a 39-amino acid linear peptide, selectively removing Lys 20 The side chain protecting group Alloc is solid-phase coupled to the side chain modifying group, and the polypeptide product is cleaved. This method has many steps, a long cycle, many impurities, and is difficult to purify.

[0004] CN110903355A discloses a solid phase method for preparing Tirzepatide, which also adopts a stepwise coupling method, wherein Lys 20 The side chain was connected by Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mono-tBu))-OH, and the 1-4 amino acids were connected by the 4-peptide fragment Boc-Tyr(tBu)-Aib-Glu(tBu)-Gly-OH, which was then cleaved to obtain the polypeptide product. Although this method selected a partial fragment coupling method, the generation of related missing peptides and racemic peptide impurities was still unavoidable.

[0005] In the prior art solid-phase synthesis of Tirzepatide, due to the large number of Tirzepatide amino acids and the large number of hydrophobic amino acids in the sequence, intramolecular hydrogen bonds are easily formed during the coupling process, resulting in severe β-folding and causing resin polycondensation. If a step-by-step coupling is performed, the difficulty of amino acid coupling will increase and the coupling efficiency will be reduced, resulting in each coupling step after polycondensation being difficult and easily producing a large number of missing peptides. If a fragment coupling is performed, only by selecting appropriate fragments and combinations of fragments can the generation of impurities such as missing peptides and racemic peptides be suppressed or reduced, thereby improving the purity and yield of the crude peptide. Summary of the Invention

[0006] In view of this, the present invention provides a method for preparing Tirzepatide to solve the problem in the prior art that intramolecular hydrogen bonds are easily formed during the synthesis of Tirzepatide, resulting in severe β-folding, causing resin polycondensation, and producing a large number of missing peptides. The method mainly comprises the following steps:

[0007] (1) Tirzepatide isopeptide resin is prepared by solid phase peptide synthesis, wherein Thr or Ser in the Tirzepatide isopeptide resin is present in the structure of formula (1).

[0008]

[0009] wherein R1 is H or CH3;

[0010] R2 is an amino protecting group;

[0011] (2) cleaving the Tirzepatide isopeptide resin obtained in step (1) to obtain a Tirzepatide isopeptide;

[0012] (3) stirring the Tirzepatide isopeptide obtained in step (2) in a neutral or basic aqueous solution to obtain Tirzepatide.

[0013] In the above preparation method, the lysine at position 20 is coupled with Fmoc-Lys(Alloc)-OH or Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH. When the lysine is Fmoc-Lys(Alloc)-OH, after the selective removal of the Alloc protecting group from the Tirzepatide main chain isopeptide resin, the side chain is modified to obtain a Tirzepatide isopeptide resin, and after cleavage, a Tirzepatide isopeptide is obtained. When the lysine is Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH, a Tirzepatide isopeptide resin can be directly synthesized, and after cleavage, a Tirzepatide isopeptide is obtained.

[0014] In the above preparation method, the coupling between the other amino acids except for the isopeptide fragments can be performed by fragment coupling or stepwise coupling. In some embodiments, dipeptide, tripeptide or pentapeptide fragments are used; in some embodiments, only stepwise coupling is used.

[0015] Unless otherwise specified, the "isopeptide" mentioned herein refers to a structure in which the hydroxyl group on the side chain of Thr or Ser in the Tirzepatide peptide sequence is isomerized to form an ester bond, i.e., Thr or Ser is in the structure of formula (1). "Isopeptide fragment" refers to a peptide fragment in which Thr or Ser is in the structure of formula (1); "Tirzepatide isopeptide resin" refers to a Tirzepatide peptide resin in which Thr or Ser is in the structure of formula (1); "Tirzepatide main chain isopeptide resin" refers to a Tirzepatide main chain peptide resin in which Thr or Ser is in the structure of formula (1).

[0016] As a preference, the Thr or Ser in the structure of formula (1) in step (1) is selected from Thr 5 , Thr 7 , Ser 8 , Ser11 、Ser 32 、Ser 33 、Ser 39 In some embodiments, the number of Thr or Ser present in the structure of formula (1) in the Tirzepatide isopeptide resin is one; in some embodiments, the number of Thr or Ser present in the structure of formula (1) in the Tirzepatide isopeptide resin is two or more, and the amino protecting groups R2 in formula (1) may be the same or different.

[0017] More preferably, the Thr or Ser present in the structure of formula (1) in step (1) is selected from Ser 11 、Ser 32 、Ser 33 One or more of .

[0018] More preferably, the Thr or Ser present in the structure of formula (1) is Ser 11 and Ser 32 .

[0019] More preferably, the Thr or Ser present in the structure of formula (1) is Ser 32 .

[0020] Preferably, in the formula (1) of step (1), R2 is selected from Cbz, Dde, Alloc, Boc, Moz, Trt, Dmb, Mmt, and Mtt.

[0021] More preferably, R2 in the formula (1) is selected from Boc and Moz.

[0022] Preferably, the pH of the neutral or alkaline aqueous solution in step (3) is 7-11.

[0023] More preferably, the pH of the neutral or alkaline aqueous solution is 7-9.

[0024] After cleavage, Tirzepatide isopeptide resin can undergo O→N migration reaction in neutral or alkaline aqueous solution to ultimately obtain Tirzepatide. The mechanism is as follows:

[0025]

[0026] The isopeptide fragment with the structure of formula (1) has excellent solubility. When used as a coupling component in polypeptide synthesis, it can effectively destroy the intramolecular hydrogen bonds of the peptide chain, causing it to form a "disordered curl" state, thereby reducing β-folding and resin condensation, greatly improving the coupling effect of hydrophobic amino acids, and ultimately reducing the production of missing peptides, thereby improving the yield and purity of the target product.

[0027] In the prior art, there are two main methods for isomerizing the hydroxyl groups on the side chains of Thr or Ser residues into esters: liquid phase method and solid phase method, as follows:

[0028] 1. Liquid phase method: The protected amino acid is reacted with 1H-benzotriazole to obtain an acylbenzotriazole intermediate, which is then reacted with the protected Ser / Thr in a DIEA-acetonitrile system to obtain an isopeptide fragment, which is then used for main chain peptide condensation. 2. Solid phase method: This can be divided into the following two methods: (1) The protected Ser / Thr is coupled to the resin, and the side chain hydroxyl of the Ser / Thr is reacted with the protected amino acid under ester condensation conditions to obtain an isopeptide fragment resin. The isopeptide fragment resin is cleaved to obtain an isopeptide fragment, which is then used for main chain peptide condensation; (2) The protected Ser / Thr is directly constructed on the main chain peptide resin under ester condensation conditions to obtain an isopeptide structure, forming a main chain isopeptide resin. In some embodiments, the synthesis of the isopeptide fragment is carried out by the liquid phase method; in some embodiments, the synthesis of the isopeptide fragment is carried out by the solid phase method.

[0029] The crude Tirzepatide prepared above was further purified and lyophilized to obtain pure Tirzepatide.

[0030] During the solid-phase synthesis of Tirzepatide, since Tirzepatide has a large number of amino acids and contains a large number of hydrophobic amino acids in the sequence, it is easy to form intramolecular hydrogen bonds, resulting in severe β-folding, causing resin polycondensation. When coupled step by step, it increases the difficulty of amino acid coupling and reduces the efficiency of coupling, resulting in each coupling step after polycondensation being difficult and easily producing a large number of missing peptides. The present invention uses the structure of formula (1) to prepare Tirzepatide. Since the isopeptide fragments present in the structure of formula (1) have excellent solubility, when used as coupling components for polypeptide synthesis, they can effectively destroy the intramolecular hydrogen bonds of the peptide chain, causing it to form a "disordered coil" state, thereby reducing β-folding and resin polycondensation, greatly improving the coupling effect of hydrophobic amino acids, ultimately reducing the production of missing peptides, and improving the yield and purity of the target product. This is a preparation method suitable for large-scale production of Tirzepatide. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Figure 1 is a flow chart of the synthesis reaction of the Boc-Ser[O(Fmoc-Pro)]-OH isopeptide fragment;

[0032] Figure 2 Figure 1 is a flow chart of the synthetic reaction of the Moz-Ser{O[Fmoc-Ser(OtBu)]}-OH isopeptide fragment;

[0033] Figure 3Figure 1 is a flow chart of the synthesis reaction of the Boc-Ser{O[Fmoc-Tyr(OtBu)]}-OH isopeptide fragment;

[0034] Figure 4 The figure is a flow chart of the synthesis reaction of the Alloc-Ser{O[Fmoc-Thr(OtBu)]}-OH isopeptide fragment;

[0035] Figure 5 The figure is a flow chart of the synthesis reaction of the Boc-Thr{O[Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly]}-OH isopeptide fragment;

[0036] Figure 6 The synthetic reaction flow chart of Boc-Thr[O-(Fmoc-Phe)]-OH isopeptide fragment. DETAILED DESCRIPTION

[0037] The present invention discloses a method for preparing Tirzepatide. Those skilled in the art may refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications obvious to those skilled in the art are considered to be included in the present invention.

[0038] In a specific embodiment of the present invention, all amino acids coupled with protecting groups are commercially available. The amino acids coupled with protecting groups in the preparation of Tirzepatide are shown in Table 1. The Chinese names corresponding to the English abbreviations involved in the present invention are shown in Table 2.

[0039] Table 1 Amino acids coupled with protecting groups during the preparation of Tirzepatide

[0040]

[0041] Table 2 Chinese names corresponding to the English abbreviations involved in the present invention

[0042]

[0043]

[0044] The reagents used in the synthesis method of Tirzepatide provided by the present invention can all be obtained commercially.

[0045] The technical solution of the present invention is further described in detail according to the following embodiments.

[0046] Example 1 Preparation of Tirzepatide 1

[0047] (1) Synthesis of Boc-Ser[O(Fmoc-Pro)]-OH isopeptide fragment

[0048] 47.6 g of BtH was weighed and dissolved in 600 mL of DCM. 14.3 g of SOCl2 was slowly added dropwise at room temperature to obtain a clear, transparent solution. 33.7 g of Fmoc-Pro-OH was added portionwise, during which a large amount of white solid was continuously produced. After the addition was completed, the reaction was continued at room temperature for 2.5 h. The filtrate was collected by suction and the filter cake was washed three times with EtOAc. The organic phases were combined and concentrated to dryness under reduced pressure. The resulting solid was redissolved in 600 mL of EtOAc, washed three times with a saturated Na2CO3 solution, dried over anhydrous MgSO4, and the solvent was evaporated to obtain 43.8 g of Fmoc-Pro-Bt intermediate in a 100% yield.

[0049] 8.76g of Fmoc-Pro-Bt was weighed and mixed with 4.10g of Boc-Ser-OH, dissolved in 200mL of acetonitrile, and 7.74g of DIEA was added. The mixture was stirred at room temperature for 12 hours. After the reaction, 1mol / L HCl was added to acidify the mixture, and the solvent was evaporated under reduced pressure. The residue was redissolved in 300mL of EtOAc, washed with 1mol / L HCl, dried over anhydrous MgSO4, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography to obtain 8.80g of the Boc-Ser[O(Fmoc-Pro)]-OH isopeptide fragment in an 84.0% yield. Figure 1 The synthetic reaction flow chart of Boc-Ser[O(Fmoc-Pro)]-OH isopeptide fragment.

[0050] (2) Preparation of Tirzepatide isopeptide resin

[0051] 5.00 g of Rink Amide MBHA resin (SD = 0.46 mmol / g, total substitution 2.30 mmol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and then drained. 50 mL of a 20% piperidine-DMF solution was added and reacted for 15 minutes to remove the Fmoc protection. The resin was drained and washed six times with DMF. The reaction was positive for ninhydrin.

[0052] 6.9 mmol Fmoc-Ser(OtBu)-OH and 1.12 g HOBt were weighed and dissolved in 50 mL DMF. 0.96 g DIC was added under ice bath and activated for 5 min. The activated reaction solution was added to a solid phase reactor and reacted for 2 h. The ninhydrin test was negative. The resin was drained and washed with DMF 6 times to obtain Fmoc-Ser(OtBu)-Rink MBHA resin.

[0053] The Fmoc-Ser(OtBu)-Rink MBHA resin obtained in the above steps was placed in a solid-phase reactor, swollen and washed with DMF, and deprotected twice with a 20% piperidine / DMF solution (v / v), the first time for 5 minutes and the second time for 15 minutes. The resin was washed six times with DMF and tested positive with ninhydrin. Fmoc-Pro-OH (2.33 g, 6.9 mmol) and HOBt (1.12 g, 8.3 mmol) were weighed and dissolved in 50 mL of DMF. DIC (1.05 g, 8.3 mmol) was added under ice-cooling and activated for 5 minutes. The activated reaction solution was added to a solid-phase reactor and reacted for 2 hours. The reaction was negative with ninhydrin, then drained and washed six times with DMF.

[0054] Repeat the above deprotection and condensation steps to continue coupling Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(OtBu)-OH, Boc-Ser[O(Fmoc-Pro)]-OH prepared in step (1), Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γGlu-Eicosanedioic tBuester)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Tyr( OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Thr(OtBu)-OH, Fmoc-Phe-OH, F moc-Thr(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Fmoc-Tyr(OtBu)-OH. After the reaction, the Fmoc protection was removed, the product was washed with DMF and DCM three times respectively, and dried to obtain 19.06 g of Tirzepatide isopeptide resin.

[0055] (3) Preparation of Tirzepatide Isopeptide

[0056] The Tirzepatide isopeptide resin prepared in step (2) was added to 190 mL of a frozen lysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% Mpr) (v / v) and allowed to react for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of ice-cold ether was added to precipitate a white precipitate. The precipitate was washed three times with ice-cold ether and dried under vacuum to yield 11.62 g of Tirzepatide isopeptide.

[0057] (4) Preparation of Tirzepatide

[0058] The Tirzepatide isopeptide prepared in step (3) was dissolved in 581 mL of pure water (pH = 7.0) to a final concentration of 20 mg / mL and stirred at room temperature to rearrange it into the target Tirzepatide. The reaction progress was monitored by HPLC. The reaction was completed after about 48 hours. The obtained product was purified by RP-HPLC, and the main peak was collected, concentrated and lyophilized to obtain 3.28 g of product with a purity of 99.1% and an overall yield of 29.63%.

[0059] Example 2 Preparation of Tirzepatide 2

[0060] (1) Synthesis of Moz-Ser{O[Fmoc-Ser(OtBu)]}-OH isopeptide fragment

[0061] 38.3 g of Fmoc-Ser(OtBu)-OH was weighed and dissolved in 600 mL of DCM. 20.6 g of DCC was added in portions under ice bath. After stirring at low temperature for 10 min, 14.3 g (120 mmol) of BtH was added and stirring was continued for 30 min. The reaction was then moved to room temperature for 12 h. After the reaction was completed, the precipitate was removed by filtration, the filter cake was washed three times with EtOAc, and the organic phases were combined and concentrated to dryness under reduced pressure. The resulting solid was redissolved in 600 mL of EtOAc, washed three times with saturated Na2CO3 solution, dried over anhydrous MgSO4, and the solvent was evaporated to obtain 47.6 g of Fmoc-Ser(OtBu)-Bt intermediate with a yield of 98.3%.

[0062] 9.68 g of Fmoc-Ser(OtBu)-Bt was weighed and mixed with 5.06 g of Moz-Ser-OH, dissolved in 200 mL of acetonitrile, and 7.74 g of DIEA was added. The mixture was stirred at room temperature for 12 hours. After the reaction, 1 mol / L HCl was added to acidify the mixture, and the solvent was evaporated under reduced pressure. The residue was redissolved in 300 mL of EtOAc, washed with 1 mol / L HCl, dried over anhydrous MgSO4, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography to obtain 9.95 g of the Moz-Ser{O[Fmoc-Ser(OtBu)]}-OH isopeptide fragment in an 80.4% yield. Figure 2Synthesis reaction flow chart for the isopeptide fragment of Moz-Ser{O[Fmoc-Ser(OtBu)]}-OH.

[0063] (2) Preparation of Tirzepatide isopeptide resin

[0064] Weigh 5.00 g Rink Amide MBHA resin (SD = 0.52 mmol / g, total substitution 2.60 mmol), add to the solid phase reactor, swell with DMF, and then dry. Add 50 mL of 20% piperidine-DMF solution to remove the Fmoc protection for 15 min, dry, and wash with DMF 6 times. The ninhydrin test is positive.

[0065] Weigh 7.8 mmol of Fmoc-Ser(OtBu)-OH, 1.26 g of HOBt, and dissolve in 50 mL of DMF. Add 1.08 g of DIC under ice bath and activate for 5 min. Add the activated reaction solution to the solid phase reactor and react for 2 h. The ninhydrin test is negative. Dry and wash with DMF 6 times to obtain Fmoc-Ser(OtBu)-Rink MBHA resin.

[0066] Add 20% piperidine / DMF solution (v / v) to the Fmoc-Ser(OtBu)-Rink MBHA resin obtained in the above step to deprotect twice, 5 min for the first time and 15 min for the second time. Wash with DMF 6 times and the ninhydrin test is positive. Weigh Fmoc-Pro-OH (2.62 g, 7.8 mmol) and HOBt (1.26 g, 9.4 mmol) and dissolve in 50 mL of DMF. Add DIC (1.18 g, 9.4 mmol) under ice bath and activate for 5 min. Add the activated reaction solution to the solid phase reactor and react for 2 h. The ninhydrin test is negative. Dry and wash with DMF 6 times.

[0067] Repeat the above deprotection and condensation steps to continue coupling Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Moz-Ser{O[Fmoc-Ser(OtBu)]}-OH prepared in step (1), Fmoc-Gly-Gly-Pro-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γGlu-Eicosanedioic tBu ester)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(O tBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Tyr(OtBu)-OH Fmoc-Asp(OtBu)-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Thr(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Fmoc-Tyr(OtBu)-OH. After the reaction, the Fmoc protection was removed, the product was washed with DMF and DCM three times respectively, and dried to obtain 21.12 g of Tirzepatide isopeptide resin.

[0068] (3) Preparation of Tirzepatide Isopeptide

[0069] The Tirzepatide isopeptide resin prepared in step (2) was added to 220 mL of a frozen lysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% Mpr) (v / v) and allowed to react for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of ice-cold ether was added to precipitate a white precipitate. The precipitate was washed three times with ice-cold ether and dried under vacuum to yield 13.32 g of Tirzepatide isopeptide.

[0070] (4) Preparation of Tirzepatide

[0071] The Tirzepatide isopeptide prepared in step (3) was dissolved in 666 mL of PBS solution (pH = 7.6) to a final concentration of 20 mg / mL, and rearranged into the target Tirzepatide at room temperature with stirring. The reaction progress was monitored by HPLC, and the reaction was completed after about 12 h. The resulting product was purified by RP-HPLC, and the main peak was collected and concentrated and lyophilized to obtain 3.96 g of product with a purity of 99.0%, and a total yield of 31.65%.

[0072] Example 3 Preparation 3 of Tirzepatide

[0073] (1) Synthesis of Moz-Ser[O(Fmoc-Pro)]-OH isopeptide fragment

[0074] Weigh 47.6 g of BtH into 600 mL of DCM, and slowly drop 14.3 g of SOCl2 into it at room temperature. After the dropwise addition is completed, a clear and transparent solution is obtained. Add 33.7 g of Fmoc-Pro-OH in batches, and a large amount of white solid is generated during the addition. After the addition is completed, continue to react at room temperature for 2.5 h. Collect the filtrate by suction filtration, and wash the filter cake with EtOAc for 3 times. Combine the organic phases, and concentrate to dryness under reduced pressure. The obtained solid is dissolved in 600 mL of EtOAc, washed with saturated Na2CO3 solution for 3 times, dried over anhydrous MgSO4, and evaporated to obtain 43.8 g of Fmoc-Pro-Bt intermediate, with a yield of 100%.

[0075] Weigh 8.76 g of Fmoc-Pro-Bt, and mix it with 5.38 g of Moz-Ser-OH to dissolve in 200 mL of acetonitrile. Add 7.74 g of DIEA, and stir the reaction at room temperature for 12 h. After the reaction is completed, acidify by adding 1 mol / L HCl, and evaporate the solvent under reduced pressure. Dissolve the residue in 300 mL of EtOAc, wash with 1 mol / L HCl, dry over anhydrous MgSO4, and evaporate the solvent under reduced pressure. Purify the residue by column chromatography to obtain 9.77 g of Moz-Ser[O(Fmoc-Pro)]-OH isopeptide fragment, with a yield of 75.4%. The synthesis reaction flow chart is similar to Figure 1 .

[0076] (2) Synthesis of Boc-Ser{O[Fmoc-Tyr(OtBu)]}-OH

[0077] Weigh 45.9 g of Fmoc-Tyr(OtBu)-OH into 600 mL of DCM, and add 20.6 g of DCC in batches under ice bath, and after stirring for 10 min at low temperature, add 14.3 g (120 mmol) of BtH, continue stirring for 30 min, and then move to room temperature for reaction for 12 h. After the reaction is completed, remove the precipitate by suction filtration, wash the filter cake with EtOAc for 3 times, combine the organic phase, and concentrate to dryness under reduced pressure. The obtained solid is dissolved in 600 mL of EtOAc, washed with saturated Na2CO3 solution for 3 times, dried over anhydrous MgSO4, and evaporate the solvent to obtain 53.7 g of Fmoc-Tyr(OtBu)-Bt intermediate, with a yield of 95.9%.

[0078] Weigh 11.20 g of Fmoc-Tyr(OtBu)-Bt, and mix with 4.10 g of Boc-Ser-OH to dissolve in 200 mL of acetonitrile, and add 7.74 g of DIEA, and stir for reaction at room temperature for 12 h. After the reaction is completed, acidify by adding 1 mol / L HCl, and evaporate the solvent under reduced pressure. Dissolve the residue in 300 mL of EtOAc, wash with 1 mol / L HCl, dry over anhydrous MgSO4, and purify the residue by column chromatography to obtain 10.61 g of Boc-Ser{O[Fmoc-Tyr(OtBu)]}-OH isopeptide fragment, with a yield of 82.04%. Figure 3 The following is a reaction flow chart for the synthesis of Boc-Ser{O[Fmoc-Tyr(OtBu)]}-OH isopeptide fragment.

[0079] (3) Preparation of Tirzepatide isopeptide resin

[0080] Weigh 10.00 g of Rink Amide MBHA resin (SD = 0.52 mmol / g, total substitution degree 5.20 mmol), and add to a solid-phase reactor, and after swelling with DMF, dry by suction. Add 100 mL of 20% piperidine-DMF solution for reaction for 15 min to remove Fmoc protection, dry by suction, wash with DMF for 6 times, and test positive by ninhydrin detection.

[0081] Weigh 15.6 mmol of Fmoc-Ser(OtBu)-OH, 2.52 g of HOBt, and dissolve in 120 mL of DMF, and add 2.16 g of DIC under ice bath, activate for 5 min, and add the activated reaction solution to the solid-phase reactor, and react for 2 h, test negative by ninhydrin detection, dry by suction, and wash with DMF for 6 times to obtain Fmoc-Ser(OtBu)-Rink MBHA resin.

[0082] The Fmoc-Ser(OtBu)-Rink MBHA resin obtained in the above steps was placed in a solid-phase reactor, swollen and washed with DMF, and deprotected twice with a 20% piperidine / DMF solution (v / v), the first time for 5 minutes and the second time for 15 minutes. The resin was washed six times with DMF and tested positive with ninhydrin. Fmoc-Pro-OH (5.26 g, 15.6 mmol) and HOBt (2.53 g, 18.7 mmol) were weighed and dissolved in 100 mL of DMF. DIC (2.36 g, 18.7 mmol) was added under ice-cooling and activated for 5 minutes. The activated reaction solution was added to a solid-phase reactor and reacted for 2 hours. The ninhydrin test was negative, the resin was drained, and washed six times with DMF.

[0083] Repeat the above deprotection and condensation steps to continue coupling Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(OtBu)-OH, Moz-Ser[O(Fmoc-Pro)]-OH prepared in step (1), Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γGlu-Eicosanedioic tBuester)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc- Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Boc-Ser{O[Fmoc-Tyr(O) prepared in step (2) tBu)]}-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Thr(OtBu)-OH, Fmoc-Phe-OH, F moc-Thr(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Fmoc-Tyr(OtBu)-OH. After the reaction, the Fmoc protection was removed, the product was washed with DMF and DCM three times respectively, and dried to obtain 40.41 g of Tirzepatide isopeptide resin.

[0084] (4) Preparation of Tirzepatide Isopeptide

[0085] The Tirzepatide isopeptide resin prepared in step (3) was added to 390 mL of ice-cold cleavage reagent 92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% Mpr (v / v) and reacted for 2 h. The reaction was filtered and the filtrate was concentrated. 2 L of ice-cold ether was added to precipitate a white solid, which was washed with ice-cold ether three times and dried under vacuum to obtain Tirzepatide isopeptide 27.11 g.

[0086] (5) Preparation of Tirzepatide

[0087] The Tirzepatide isopeptide prepared in step (4) was dissolved in 1355 mL of PBS solution (pH = 8.0) to a final concentration of 20 mg / mL, and rearranged into the target Tirzepatide at room temperature under stirring. The reaction progress was monitored by HPLC, and the reaction was completed after about 12 h. The obtained product was purified by RP-HPLC, and the main peak was collected and concentrated and lyophilized to obtain 7.86 g of product with a purity of 99.4% and a total yield of 31.41%.

[0088] Example 4 Preparation 4 of Tirzepatide

[0089] (1) Synthesis of Alloc-Ser{O[Fmoc-Thr(OtBu)]}-OH fragment

[0090] Fmoc-Thr(OtBu)-OH 39.7 g was weighed into 600 mL of DCM, and 20.6 g of DCC was added portionwise under ice bath. After stirring at low temperature for 10 min, 14.3 g (120 mmol) of BtH was added, and stirring was continued for 30 min, followed by reaction at room temperature for 12 h. After the reaction was completed, the precipitate was removed by suction filtration, and the filter cake was washed with EtOAc three times. The organic phase was combined and concentrated under reduced pressure to dryness. The obtained solid was dissolved in 600 mL of EtOAc, washed with saturated Na2CO3 solution three times, dried over anhydrous MgSO4, and the solvent was evaporated to obtain 47.4 g of Fmoc-Thr(OtBu)-Bt intermediate with a yield of 95.2%.

[0091] Fmoc-Thr(OtBu)-Bt 10.00 g was weighed, mixed with 3.78 g of Alloc-Ser-OH, and dissolved in 200 mL of acetonitrile, and 7.74 g of DIEA was added. The reaction was stirred at room temperature for 12 h. After the reaction was completed, it was acidified by adding 1 mol / L HCl, and the solvent was evaporated under reduced pressure. The residue was dissolved in 300 mL of EtOAc, washed with 1 mol / L HCl, dried over anhydrous MgSO4, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography to obtain 8.06 g of Alloc-Ser{O[Fmoc-Thr(OtBu)]}-OH isopeptide fragment with a yield of 71.0%. Figure 4The synthetic reaction flow chart of the Alloc-Ser{O[Fmoc-Thr(OtBu)]}-OH isopeptide fragment.

[0092] (2) Preparation of Tirzepatide isopeptide resin

[0093] Weigh 10.00 g of Ramaga MBHA resin (SD = 0.4 mmol / g, total substitution 4.0 mmol) into a solid-phase reactor, swell with DMF, and drain. Add 100 mL of 20% piperidine-DMF solution and react for 15 minutes to remove the Fmoc protection. Drain the solution and wash six times with DMF. Ninhydrin test results are positive.

[0094] 12.0 mmol Fmoc-Ser(OtBu)-OH and 1.94 g HOBt were weighed and dissolved in 100 mL DMF. 1.81 g DIC was added under ice bath and activated for 5 min. The activated reaction solution was added to a solid phase reactor and reacted for 2 h. The ninhydrin test was negative. The resin was drained and washed with DMF 6 times to obtain Fmoc-Ser(OtBu)-Rink MBHA resin.

[0095] The Fmoc-Ser(OtBu)-Rink MBHA resin obtained in the above steps was placed in a solid-phase reactor, swollen and washed with DMF, and deprotected twice with a 20% piperidine / DMF solution (v / v), the first time for 5 minutes and the second time for 15 minutes. The resin was washed six times with DMF and tested positive with ninhydrin. Fmoc-Pro-OH (4.04 g, 12 mmol) and HOBt (1.94 g, 14.4 mmol) were weighed and dissolved in 100 mL of DMF. DIC (1.81 g, 14.4 mmol) was added under ice-cooling and activated for 5 minutes. The activated reaction solution was added to a solid-phase reactor and reacted for 2 hours. The reaction was negative with ninhydrin, then drained and washed six times with DMF.

[0096] Repeat the above steps to continue coupling Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(OtBu)-OH, Boc-Ser[O(Fmoc-Pro)]-OH prepared in step (1) of Example 1, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γGlu-Eicosanedioic tBu ester)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Tyr(OtBu) )-OH, Fmoc-Asp(OtBu)-OH, Alloc-Ser{O[Fmoc-Thr(OtBu)]}-OH prepared in step (1), Fmoc-Phe-OH, F moc-Thr(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Fmoc-Tyr(OtBu)-OH. After the reaction, 4 mmol Pd(PPh3)4 and 400 mmol phenylsilane were weighed and dissolved in 300 mL DCM, added to the resin, and stirred at room temperature for 2 h. After the reaction was completed, the resin was drained and washed with DMF three times to remove the N-terminal Fmoc protecting group to obtain 34.77 g of Tirzepatide isopeptide resin.

[0097] (3) Preparation of Tirzepatide Isopeptide

[0098] The Tirzepatide isopeptide resin prepared in step (2) was added to 350 mL of a frozen lysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% Mpr) (v / v) and allowed to react for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of ice-cold ether was added to precipitate a white precipitate. The precipitate was washed three times with ice-cold ether and dried under vacuum to yield 20.06 g of Tirzepatide isopeptide.

[0099] (4) Preparation of Tirzepatide

[0100] The Tirzepatide isopeptide prepared in step (3) was dissolved in 1003 mL of PBS buffer solution (pH = 7.4) to a final concentration of 20 mg / mL, and rearranged into the target Tirzepatide at room temperature with stirring. The reaction progress was monitored by HPLC, and the reaction was completed after about 12 h. The resulting product was purified by RP-HPLC, and the main peak was collected and concentrated for lyophilization to obtain 5.66 g of product with a purity of 99.2%, and a total yield of 29.40%.

[0101] Example 5 Preparation 5 of Tirzepatide

[0102] (1) Synthesis of Boc-Thr{O[Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly]}-OH fragment

[0103] 1.1 Synthesis of Boc-Thr-2-CTC resin

[0104] Boc-Thr-OH (13.10 g, 60 mmol) was weighed into 300 mL of DCM, and after the addition of DIEA (19.35 g, 150 mmol), it was dissolved at room temperature. The mixture was added to the resin, and the reaction was carried out at room temperature for 2 h. After the reaction was completed, 10 mL of methanol was added, and the mixture was sealed for 1 h, then dried, washed with DMF and DCM three times each, and dried in vacuum to obtain Boc-Thr-2-CTC resin with a weight of 35.07 g and a substitution degree of 0.79 mmol / g.

[0105] 1.2 Synthesis of Boc-Thr[O(Fmoc-Gly)]-2-CTC resin

[0106] Boc-Thr-2-CTC resin (10 mmol) prepared in step 1.1 was weighed into a solid-phase reactor, and after swelling with DCM, it was dried. Fmoc-Gly-OH (8.92 g) was dissolved in a mixture of 120 mL of DCM and 10 mL of DMF, and after the addition of DIC (4.54 g) and DMAP (370 mg) in an ice bath, the mixture was stirred for 30 min. The reaction solution was added to the Boc-Thr-2-CTC resin, and the reaction was carried out at room temperature for 12 h. After the reaction was completed, the mixture was dried, washed with DMF and DCM three times each, and dried in vacuum to obtain Boc-Thr[O(Fmoc-Gly)]-2-CTC resin with a weight of 15.46 g and a yield of 100%.

[0107] 1.3 Synthesis of Boc-Thr{O[Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly]}-2-CTC resin

[0108] Place the Boc-Thr[O(Fmoc-Gly)]-2-CTC resin prepared in step 1.2 in a solid-phase reactor, add DMF to swell it, and then drain it. Add 150 mL of 20% piperidine-DMF solution and react for 15 minutes to remove the Fmoc protection. Wash it with DMF six times. Then, follow the coupling steps to couple Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, and Fmoc-Tyr(OtBu)-OH. After the coupling is completed, wash it with DMF and DCM three times each, drain it, and set aside.

[0109] 1.4 Synthesis of Boc-Thr{O[Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly]}-OH

[0110] The Boc-Thr{O[Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly]}-2-CTC resin prepared in step 1.3 was added to 200 mL of 20% TFE / 80% DCM (v:v) lysis buffer and stirred at room temperature for 2 h. After the reaction, the filtrate was collected by suction and the resin was washed with DCM. The combined filtrates were concentrated under reduced pressure to yield 9.67 g of a foamy solid in a 97.9% yield. Figure 5 The synthetic reaction flow chart of Boc-Thr{O[Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly]}-OH isopeptide fragment.

[0111] (2) Synthesis of Tirzepatide Isopeptide Resin

[0112] Weigh 10.00 g of Ramaga MBHA resin (SD = 0.40 mmol / g, total substitution 4.0 mmol) and add it to a solid-phase reactor. Swell it with DMF and drain it. Add 100 mL of 20% piperidine-DMF solution and react for 15 minutes to remove the Fmoc protection. Drain it and wash it six times with DMF. The reaction is positive for ninhydrin.

[0113] 12.0 mmol Fmoc-Ser(OtBu)-OH and 2.02 g HOBt were weighed and dissolved in 100 mL DMF. 1.90 g DIC was added under ice bath and activated for 5 min. The activated reaction solution was added to a solid phase reactor and reacted for 2 h. The ninhydrin test was negative. The resin was drained and washed with DMF 6 times to obtain Fmoc-Ser(OtBu)-Rink MBHA resin.

[0114] The Fmoc-Ser(OtBu)-Rink MBHA resin obtained in the above steps was placed in a solid-phase reactor, swollen and washed with DMF, and deprotected twice with a 20% piperidine / DMF solution (v / v), the first time for 5 minutes and the second time for 15 minutes. The resin was washed six times with DMF and tested positive with ninhydrin. Fmoc-Pro-OH (4.04 g, 12 mmol) and HOBt (1.94 g, 14.4 mmol) were weighed and dissolved in 100 mL of DMF. DIC (1.81 g, 14.4 mmol) was added under ice-cooling and activated for 5 minutes. The activated reaction solution was added to a solid-phase reactor and reacted for 2 hours. The reaction was negative with ninhydrin, then drained and washed six times with DMF.

[0115] Repeat the above deprotection and condensation steps to continue coupling Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(OtBu)-OH, Boc-Ser[O(Fmoc-Pro)]-OH prepared in step (1) of Example 1, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γGlu-Eicosanedioic tBu ester)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Boc-Ser{O[Fmoc-Tyr(OtBu)]}-OH prepared in step (2) of Example 3, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Thr(OtBu)-OH, Fmoc-Phe-OH, Boc-Thr{O[Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly]}-OH prepared in step (1). After the reaction, the Boc protection was removed, the product was washed with DMF and DCM three times respectively, and dried to obtain 34.88 g of Tirzepatide isopeptide resin.

[0116] (3) Preparation of Tirzepatide Isopeptide

[0117] The Tirzepatide isopeptide resin prepared in step (2) was added to 350 mL of a frozen lysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% Mpr) (v / v) and allowed to react for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of ice-cold ether was added to precipitate a white precipitate. The precipitate was washed three times with ice-cold ether and dried under vacuum to yield 11.62 g of Tirzepatide isopeptide.

[0118] (4) Preparation of Tirzepatide

[0119] The Tirzepatide isopeptide prepared in step (3) was dissolved in 581 mL of PBS buffer solution (pH = 7.4) to a final concentration of 20 mg / mL and stirred at room temperature to rearrange the target Tirzepatide. The reaction progress was monitored by HPLC. The reaction was complete after 12 h. The obtained product was purified by RP-HPLC, and the main peak was collected, concentrated and lyophilized to obtain 6.07 g of product with a purity of 99.3% and an overall yield of 31.53%.

[0120] Example 6 Preparation of Tirzepatide

[0121] (1) Synthesis of Boc-Thr[O-(Fmoc-Phe)]-OH fragment

[0122] 47.6 g of BtH was weighed and dissolved in 600 mL of DCM. 14.3 g of SOCl2 was slowly added dropwise at room temperature to obtain a clear and transparent solution. 38.7 g of Fmoc-Phe-OH was added in batches, during which a large amount of white solid was continuously produced. After the addition was completed, the reaction was continued at room temperature for 2.5 h. The filtrate was collected by suction and the filter cake was washed three times with EtOAc. The organic phases were combined and concentrated to dryness under reduced pressure. The resulting solid was redissolved in 600 mL of EtOAc, washed three times with a saturated Na2CO3 solution, dried over anhydrous MgSO4, and the solvent was evaporated to obtain 48.8 g of Fmoc-Phe-Bt intermediate with a yield of 100%.

[0123] 9.76g of Fmoc-Phe-Bt was weighed, mixed with 4.38g of Boc-Thr-OH, dissolved in 200mL of acetonitrile, and 7.74g of DIEA was added. The reaction was stirred at room temperature for 12 hours. After completion of the reaction, 1mol / L HCl was added to acidify the mixture, and the solvent was evaporated under reduced pressure. The residue was redissolved in 300mL of EtOAc, washed with 1mol / L HCl, dried over anhydrous MgSO4, and purified by column chromatography to obtain 9.96g of the Boc-Thr[O-(Fmoc-Phe)]-OH isopeptide fragment in an 84.6% yield. Figure 6 The synthetic reaction flow chart of Boc-Thr[O-(Fmoc-Phe)]-OH isopeptide fragment.

[0124] (2) Synthesis of Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly-Thr(OtBu)-OH fragment

[0125] 2.1 Preparation of Fmoc-Thr(OtBu)-2-CTC resin

[0126] 30 g of 2-CTC resin (SD = 1.17 mmol / g, total substitution degree 35.1 mmol) was weighed and added to a solid phase reactor, swollen with DMF, and then dried. Fmoc-Thr(OtBu)-OH (23.86 g, 60 mmol) was weighed and suspended in 300 mL of DCM. DIEA (19.35 g, 150 mmol) was added and dissolved at room temperature. The mixed solution was added to the resin and reacted at room temperature for 2 h. After the reaction, 10 mL of methanol was added, the mixture was sealed for 1 h, dried, washed three times with DMF and DCM respectively, and dried under vacuum to obtain Fmoc-Thr(OtBu)-2-CTC resin weighing 40.07 g and a substitution degree of 0.70 mmol / g.

[0127] 2.2 Preparation of Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly-Thr(OtBu)-2-CTC resin

[0128] Weigh 14.29 g of the Fmoc-Thr(OtBu)-2-CTC resin (10 mmol) prepared in step 2.1 into a solid-phase reactor. Add DMF to swell the resin and drain. Then add 150 mL of a 20% piperidine-DMF solution and react at room temperature for 15 minutes to remove the Fmoc protection. The resin is then washed six times with DMF and drained for later use. Weigh 8.92 g of Fmoc-Gly-OH and 4.86 g of HOBt and dissolve them in 100 mL of DMF. Add 4.54 g of DIC in an ice bath and activate for 30 minutes. The reaction mixture is then added to the resin and allowed to react at room temperature for 2 hours. After the reaction was completed, the ninhydrin test was negative, the resin was drained, and the resin was washed 6 times with DMF and entered the next coupling cycle, in which Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, and Fmoc-Tyr(OtBu)-OH were coupled in sequence. After the reaction was completed, the resin was drained, washed 3 times with DMF and DCM, respectively, and dried in vacuo to obtain 23.37 g of Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly-Thr(OtBu)-2-CTC resin with a yield of 100%.

[0129] 2.3 Preparation of Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly-Thr(OtBu) fragment

[0130] The Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly-Thr(OtBu)-2-CTC resin prepared in step 2.2 was added to 200 mL of 20% TFE / 80% DCM (v:v) cleavage solution and stirred at room temperature for 2 h. After the reaction was completed, the filtrate was collected by suction filtration and the resin was washed with DCM. The filtrate was combined and concentrated under reduced pressure to obtain Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly-Thr(OtBu), 9.02 g in weight, with a yield of 99.3%.

[0131] (3) Preparation of Tirzepatide Isopeptide Resin

[0132] Rink Amide MBHA resin (SD = 0.52 mmol / g, total substitution 5.20 mmol) was weighed, 10.00 g, into a solid phase reactor and swelled with DMF before being suctioned dry. 100 mL of 20% piperidine-DMF solution was added to remove the Fmoc protection for 15 min, suctioned dry, and washed with DMF for 6 times. The ninhydrin test was positive.

[0133] Boc-Ser[O(Fmoc-Pro)]-OH prepared in step (1) of Example 1, 15.6 mmol, 2.52 g of HOBt, were dissolved in 120 mL of DMF, and 2.16 g of DIC was added under ice bath for 5 min of activation. The activated reaction solution was added to the solid phase reactor and reacted for 2 h. The ninhydrin test was negative. The reaction was suctioned dry and washed with DMF for 6 times to obtain Boc-Ser[O(Fmoc-Pro)]-Rink MBHA resin.

[0134] The Boc-Ser[O(Fmoc-Pro)]-Rink MBHA resin obtained in the above step was placed in a solid phase reactor and swelled with DMF. The 20% piperidine / DMF solution (v / v) was used for deprotection twice, 5 min for the first time and 15 min for the second time. The resin was washed with DMF for 6 times and the ninhydrin test was positive. Fmoc-Pro-OH (5.26 g, 15.6 mmol) and HOBt (2.52 g, 18.7 mmol) were dissolved in 10 mL of DMF, and DIC (2.36 g, 18.7 mmol) was added under ice bath for 5 min of activation. The activated reaction solution was added to the solid phase reactor and reacted for 2 h. The ninhydrin test was negative. The reaction was suctioned dry and washed with DMF for 6 times.

[0135] Repeat the above deprotection and condensation steps to continue coupling Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Boc-Ser{O[Fmoc-Ser(OtBu)]}-OH prepared in step (1) of Example 2, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Gln(Trt )-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Tyr(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(OtBu)-OH, Boc-Thr[O-(Fmoc-Phe)]-OH prepared in step (1), and Fmoc-Tyr(OtBu)-Aib-Glu(OtBu)-Gly-Thr(OtBu)-OH prepared in step (2). After the reaction, 5 mmol of Pd(PPh3)4 and 500 mmol of phenylsilane were dissolved in 300 mL of DCM and added to the resin. The mixture was stirred at room temperature for 2 h. After the reaction, the Tirzepatide main chain peptide resin was filtered to obtain the resin and washed with DMF three times. 8.94 g of tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu (10.4 mmol) and 1.68 g of HOBt (12.5 mmol) were mixed and dissolved in 100 mL of DMF. After dissolution, the mixture was cooled at low temperature for 30 min, 1.57 g of DIC (12.5 mmol) was added, and the mixture was activated at low temperature for 10 min. The activated solution was added to the Tirzepatide main chain peptide resin and reacted for 2 h. The ninhydrin test was negative, the resin was dried, washed with DMF three times, and the N-terminal Fmoc protecting group was removed to obtain 42.34 g of Tirzepatide isopeptide resin.

[0136] (4) Preparation of Tirzepatide Isopeptide

[0137] The Tirzepatide isopeptide prepared in step (3) was added to 390 mL of cold cleavage reagent 92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% Mpr (v / v) and reacted for 2 h. Filtration, concentration of the filtrate, addition of 2 L of ice-cold ether, precipitation of a white solid, washing of the precipitate with ice-cold ether 3 times, and drying under vacuum gave Tirzepatide isopeptide 27.16 g.

[0138] (5) Preparation of Tirzepatide

[0139] The Tirzepatide isopeptide prepared in step (4) was dissolved in 1358 mL of Gly-NaOH buffer solution (pH = 9.0) to give a final concentration of 20 mg / mL, and rearranged into the target Tirzepatide at room temperature with stirring. The progress of the reaction was monitored by HPLC, and the reaction was completed after 12 h. The product was purified by RP-HPLC, and the main peak was collected and concentrated and lyophilized to give 7.86 g of product with a purity of 99.5% and a total yield of 31.41%.

[0140] Example 7 Preparation of Tirzepatide 7

[0141] (1) Preparation of Boc-Ser-Rink Amide MBHA Resin

[0142] 5.00 g of Rink Amide MBHA resin (SD = 0.46 mmol / g, total substitution 2.30 mmol) was weighed into a solid-phase reactor, swelled with DMF, and then drained. 50 mL of 20% piperidine-DMF solution was added to remove the Fmoc protection for 15 min, drained, and washed with DMF 6 times, and ninhydrin detection was positive.

[0143] 6.9 mmol of Boc-Ser-OH and 1.12 g of HOBt were dissolved in 50 mL of DMF, and 0.96 g of DIC was added under ice bath for 5 min of activation. The activated reaction solution was added to the solid-phase reactor, and reacted for 2 h, with ninhydrin detection being negative. It was drained, washed with DMF 6 times, and Boc-Ser-Rink MBHA resin was obtained.

[0144] (2) Preparation of Boc-Ser[O(Fmoc-Pro)]-Rink Amide MBHA Resin.

[0145] The Boc-Ser-Rink MBHA resin obtained in step (1) was placed in a solid phase reactor and swelled with DCM for three times. Fmoc-Pro-OH (2.33 g, 6.9 mmol) was weighed and dissolved in 40 mL of DCM and 10 mL of DMF mixed solvent, and DIC (1.05 g, 8.3 mmol) was added under ice bath for 5 min. The activated reaction solution was added to the solid phase reactor, and a catalytic amount of DMAP (84 mg, 0.69 mmol) was added, and the reaction was carried out at room temperature for 12 h. After being dried, it was washed with DMF and DCM for three times, and dried under vacuum to obtain Boc-Ser[O(Fmoc-Pro)]-Rink Amide MBHA resin 6.17 g, and the substitution degree was measured to be 0.37 mmol / g.

[0146] (3) Preparation of Tirzepatide isopeptide resin

[0147] The Boc-Ser[O(Fmoc-Pro)]-Rink MBHA resin obtained in step (2) was placed in a solid phase reactor and swelled with DMF for washing, and deprotected with 20% piperidine / DMF solution (v / v) twice, 5 min for the first time and 15 min for the second time, and washed with DMF for 6 times, and the ninhydrin test was positive. Fmoc-Pro-OH (5.26 g, 15.6 mmol) and HOBt (2.52 g, 18.7 mmol) were weighed and dissolved in 10 mL of DMF, and DIC (2.36 g, 18.7 mmol) was added under ice bath for 5 min. The activated reaction solution was added to the solid phase reactor, and the reaction was carried out for 2 h, and the ninhydrin test was negative, and dried, and washed with DMF for 6 times.

[0148] The above deprotection and condensation steps were repeated to continue coupling Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γGlu-Eicosanedioic tBuester)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Tyr(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Thr(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Fmoc-Tyr(OtBu)-OH. After the reaction was completed, the Fmoc protection was removed, and the resin was washed with DMF and DCM for 3 times, respectively, and dried to obtain Tirzepatide isopeptide resin 19.14 g.

[0149] (4) Preparation of Tirzepatide isopeptide

[0150] The Tirzepatide isopeptide resin prepared in step (3) was added to 190 mL of cold cleavage reagent 92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% Mpr (v / v) and reacted for 2 h. Filtration, concentration of the filtrate, addition of 2 L of ice-cold ether, precipitation of a white precipitate, washing of the precipitate with ice-cold ether for 3 times, and vacuum drying gave Tirzepatide isopeptide 11.24 g.

[0151] (5) Preparation of Tirzepatide

[0152] The Tirzepatide isopeptide prepared in step (4) was dissolved in 562 mL of Gly-NaOH buffer solution (pH = 11.0) to a final concentration of 20 mg / mL and stirred at room temperature to rearrange the target Tirzepatide. The reaction progress was monitored by HPLC. The reaction was complete after about 12 h. The obtained product was purified by RP-HPLC, and the main peak was collected, concentrated and lyophilized to obtain 3.16 g of product with a purity of 99.2% and an overall yield of 28.54%.

Claims

1. A method for preparing Tirzepatide, characterized in that: The main steps include: (1) Tirzepatide isopeptide resin was prepared by solid phase peptide synthesis, wherein Ser in Tirzepatide isopeptide resin exists in the structure of formula (1) Wherein, R1 is H; R2 is an amino protecting group; (2) cleaving the Tirzepatide isopeptide resin obtained in step (1) to obtain the Tirzepatide isopeptide; (3) stirring the Tirzepatide isopeptide obtained in step (2) in a neutral or alkaline aqueous solution to obtain Tirzepatide; Wherein, the Ser present in the structure of formula (1) is selected from Ser 11 、Ser 32 、Ser 33 One or more of; Among them, when Tirzepatide isopeptide resin is synthesized, Ser 11 Selected from Boc-Ser{O[Fmoc-Tyr(OtBu)]}-OH, Ser 32 Selected from Boc-Ser[O(Fmoc-Pro)]-OH, Moz-Ser[O(Fmoc-Pro)]-OH, Ser 33 Selected from Moz-Ser{O[Fmoc-Ser(OtBu)]}-OH.

2. The preparation method according to claim 1, characterized in that The Ser present in the structure of formula (1) is Ser 11 and Ser 32 .

3. The preparation method according to claim 1, characterized in that: The Ser present in the structure of formula (1) is Ser 32 .

4. The preparation method according to claim 1, characterized in that The pH of the neutral or alkaline aqueous solution in step (3) is 7-11.

5. The preparation method according to claim 4, characterized in that: The pH of the neutral or alkaline aqueous solution is 7-9.

6. The preparation method according to claim 1, characterized in that: The prepared Tirzepatide is further purified and lyophilized to obtain pure Tirzepatide.

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