A method for synthesizing tirzepatide

Abstract

The present invention discloses a method for synthesizing tirzepatide, belonging to the technical field of chemical pharmacy. Using a twenty-two peptide chain as a raw material, a Boc-protected twenty-two peptide chain (Boc-22AA) is obtained by biosynthesis. After activation, Boc-22AA reacts with serinamide to obtain (Boc-23AA), and deprotection of Boc gives 23-AA amide. The side chain reacts with the amino group at the ε-position of lysine in 23-AA amide to obtain 23-AA amide with a side chain (SC-23AA amide). SC-23AA amide reacts with a protected sixteen peptide active ester to obtain a tirzepatide precursor, and the tirzepatide precursor is cleaved and deprotected to obtain tirzepatide. Based on the twenty-two peptide, the total reaction yield is about 40%, and the HPLC purity is ≥99%. The synthesis method provided by the present invention has the advantages of simple operation, high yield, and high purity.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a method for synthesizing tirzepatide. Background Art

[0002] Tirzepatide is a novel dual receptor agonist of glucagon-like peptide-1 receptor (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP, also known as gastric inhibitory polypeptide), and is administered once a week.

[0003] Currently, the methods for preparing tirzepatide mainly include solid-phase synthesis methods. For example, the invention application with the application number 202211648825.5 discloses a solid-phase synthesis method for tirzepatide. By cleaving the fully protected peptide of fragment 22-29 and then connecting it to the peptide resin of fragment 30-39 by solid-phase synthesis, the peptide resin of fragment 22-39 is synthesized. In this way, impurities can be excluded from affecting subsequent reactions by washing with DMF and discarding the liquid phase. Then, it is coupled to position 15 one by one to obtain the peptide resin of fragment 15-39. After cleaving the fully protected peptide of fragment 1-14 and connecting it to fragment 15-39 by solid-phase synthesis, tirzepatide peptide resin is synthesized. The crude peptide obtained by this method has a purity of 64.87%. Another example is the invention application with the application number 202410864809.2, which discloses a method for preparing tirzepatide. It includes separately preparing the peptide resin of coupled amino acid derivatives 15-39AA, the fragment of R1-(3-14AA)-OH or the fragment of R1-(5-14AA)-OH; then sequentially coupling R1-(3-14AA)-OH and R1-Tyr(R2)-Aib-OH to the peptide resin of coupled amino acid derivatives 15-39AA by solid-phase synthesis, or sequentially coupling R1-(5-14AA)-OH, R1-Gly-OH, R1-Glu(R2)-OH, and R1-Tyr(R2)-Aib-OH to the peptide resin of coupled amino acid derivatives 15-39AA by solid-phase synthesis. Finally, the peptide resin and protecting groups are cleaved to obtain tirzepatide.

[0004] The above-mentioned existing technologies all prepare tirzepatide by solid-phase synthesis. Solid-phase synthesis requires a large excess of amino acids and condensing agents, and it is difficult to purify in the intermediate process; there are problems such as diastereoisomers generated due to amino acid racemization, deletion peptides generated due to some amino acids not being connected, and cleavage peptides generated due to peptide bond cleavage. The reaction steps are long and the preparation cost is high. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for synthesizing tirzepatide, which has the advantages of simple operation, high yield, and high purity.

[0006] The technical solution adopted by the present invention to solve the technical problem is: a method for synthesizing tirzepatide, comprising the following consecutive steps:

[0007] Step 1): Using a twenty-two peptide chain as a raw material, in a solvent, first obtain a Boc-protected twenty-two peptide chain (Boc-22AA) through biosynthesis. After activating the Boc-protected twenty-two peptide chain (Boc-22AA), react it with serinamide to obtain a Boc-protected twenty-three peptide chain (Boc-23AA), and deprotect Boc to obtain 23-AA amide;

[0008]

[0009] Step 2): In a solvent, react a side-chain active ester with the 23-AA amide prepared in Step 1) at the lysine ε-position amino group to obtain a 23-AA amide with a side chain (SC-23AA amide);

[0010]

[0011]

[0012] Step 3): In a solvent, react the 23-AA amide with a side chain (SC-23AA amide) prepared in Step 2) with a protected sixteen-peptide active ester to obtain a tirzepatide precursor;

[0013]

[0014] Step 4): The tirzepatide precursor is cleaved and deprotected to obtain a crude product of tirzepatide; after purification, tirzepatide is obtained.

[0015]

[0016] Further, in Step 1), the molar ratio of the twenty-two peptide chain to (Boc)2O is 1:2; the molar ratio of the Boc-protected twenty-two peptide chain (Boc-22AA) to serinamide is 1:1 to 1:2.

[0017] Further, in Step 2), the molar ratio of the 23-AA amide to the side-chain active ester is 1:1 to 1:1.5.

[0018] Further, in Step 3), the molar ratio of the 23-AA amide with a side chain (SC-23AA amide) to the protected sixteen-peptide active ester is 1:1 to 1:1.5.

[0019] Further, the R substitution group and the R1 substitution group in the protected hexadecapeptide active ester and the side-chain active ester both include any one of succinimide (Su), pentafluorophenyl (PFP), phthalimide (PHT), and N-5-norbornene-2,3-dicarboximide (NB).

[0020] Further, the solvent is one or more of DMF, acetonitrile, dioxane, DMSO, and DMAC.

[0021] Further, in each reaction step, the pH of the reaction system is adjusted by adding a base, and the base used is one or more of triethylamine, diisopropylethylamine, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, and lithium hydroxide.

[0022] Further, the mobile phase used in the purification process is alcohol and water.

[0023] The beneficial effects of the present invention are as follows: Compared with the prior art, the synthesis method of tirzepatide provided by the present invention directly uses 22 amino acid polypeptides in the main chain from biosynthesis, reducing the reaction steps and the racemization of chiral amino acids; reducing the research on reaction intermediate control and related substances; and significantly reducing the overall cost. In addition, the present invention uses a liquid-phase synthesis method, and each step can be directionally transformed, which can greatly improve the purity and yield and meet the strict quality control requirements for intermediates and APIs in drug research and development; moreover, the liquid-phase synthesis is not limited by the feeding scale, solving the problems of being limited by the feeding scale and being difficult to scale up in solid-phase synthesis. Description of the Drawings

[0024] Figure 1 It is the full molecular weight primary mass spectrometry diagram of the tirzepatide prepared in Example 1.

[0025] Figure 2 It is the HPLC diagram of the tirzepatide prepared in Example 1. Detailed Embodiments

[0026] The present invention will be further illustrated by specific examples below. However, these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The raw material 22-peptide used in each example is from in-house biosynthesis of the company (purity 97%). The specific synthesis method of the 22-peptide refers to patents CN118240059A, CN115991742A, CN116854805A, and CN117106055A. Currently, the synthesis of side-chain active esters is relatively mature, and the solid-phase synthesis or liquid-phase synthesis of polypeptides is also relatively mature. The side-chain active esters and the hexadecapeptide with a protecting group are from the market (purity 97%).

[0027] Example 1

[0028] At 10 - 25 °C, add 2.12 g (0.001 mol) of the 22 - peptide (22 - AA) into a 50 mL two - necked flask, add 20 mL of DMF, stir, add 0.44 g (0.002 mol) of Boc anhydride, and continue stirring for 1 - 2 h. Add it into pure water, precipitate solid, wash with water twice, and freeze - dry to remove water, obtaining Boc - protected 22 - peptide (Boc - 22AA).

[0029] At 10 - 25 °C, add the obtained Boc - 22AA into a 50 mL two - necked flask, add 25 mL of DMF, add 0.12 g (0.001 mol) of HOSU, add 0.12 g (0.001 mol) of serine amide, add 0.25 g (0.0012 mol) of DCC, adjust the pH to 9.5 - 10.5. Continue stirring for 2 - 3 h. Filter, add the filtrate into pure water, precipitate solid, wash with water twice, and freeze - dry to obtain Boc - 23AA. Add Boc - 23AA into the reaction flask, add 2N dilute hydrochloric acid, stir for 1 h, precipitate solid, filter by suction, and dry to obtain de - protected 23AA amide.

[0030] At 10 - 25 °C, add 0.92 g (0.001 mol) of side - chain active ester (OSU) into a 50 mL three - necked flask, add 20 mL of DMF, stir, add the above - obtained 23AA amide, adjust the pH to about 9.5, continue reacting for 2 h, pour it into pure water, precipitate solid, wash with water twice, and freeze - dry to obtain side - chain - bearing 23 - AA amide (SC - 23AA amide).

[0031] At 10 - 25 °C, add 2.6 g (0.001 mol) of protected 16 - peptide (16 - AA) into a 50 mL three - necked flask, add 20 mL of DMF, stir, add 0.12 g (0.001 mol) of HOSU, add the above - obtained SC - 23AA amide, add 0.25 g (0.0012 mol) of DCC, control the pH at 8.0 - 8.5, stir and react for 2 - 3 h. Filter, pour the filtrate into pure water, precipitate solid, wash with water twice to obtain the telotristat precursor.

[0032] At 10 - 25 °C, add the telotristat precursor into a 50 mL three - necked flask, add 25 mL of trifluoroacetic acid to the reaction system, stir for 1 h. Add methyl tert - butyl ether, precipitate solid, and centrifuge to obtain the crude telotristat.

[0033] The crude telotristat is prepared by preparative liquid chromatography with an ethanol / water system to obtain 2.01 g of telotristat, with a yield of 42%. The HPLC purity is greater than 99%. As Figure 1As shown, MS: m / z = 4813.7718. According to the isotope peaks and weighted average molecular weight calculation (as shown in Table 1 below), the measured isotope molecular weight of the prepared tirzepatide is 4810.4696 Da, and the measured average molecular weight is 4813.7718, both of which are consistent with the theory.

[0034] Table 1. Calculation of weighted average molecular weight

[0035]

[0036] Example 2

[0037] At 10 - 25 °C, add 2.12 g (0.001 mol) of the twenty-two peptide (22-AA) into a 50 mL two-necked flask, add 20 mL of DMF, stir, add 0.44 g (0.002 mol) of Boc anhydride, and continue stirring for 1 - 2 h. Add it to pure water, precipitate a solid, wash it twice with water, and freeze-dry to remove the moisture to obtain Boc-protected twenty-two peptide (Boc-22AA).

[0038] At 10 - 25 °C, add the obtained Boc-22AA into a 50 mL two-necked flask, add 25 mL of DMF, add 0.16 g (0.001 mol) of HONB (N-hydroxy-5-norbornene-2,3-dicarboximide), add 0.12 g (0.001 mol) of serinamide, add 0.25 g (0.0012 mol) of DCC, adjust the pH to 9.5 - 10.5, and continue stirring for 2 - 3 h. Filter, add the filtrate to pure water, precipitate a solid, wash it twice with water, and freeze-dry to obtain Boc-23AA. Add 2N dilute hydrochloric acid, stir for 1 h, precipitate a solid, filter by suction, and dry to obtain deprotected 23AA amide.

[0039] At 10 - 25 °C, add 0.92 g (0.001 mol) of the side-chain active ester (OSU) into a 50 mL three-necked flask, add 20 mL of DMF, stir, add the above-obtained 23AA amide, adjust the pH to 8.0 - 9.0, continue the reaction for 2 h, pour it into pure water, precipitate a solid, wash it twice with water, and freeze-dry to obtain the side-chain-containing 23-AA amide (SC-23AA amide).

[0040] At 10 - 25 °C, add 2.6 g (0.001 mol) of the protected hexadecapeptide (16 - AA) with protection to a 50 mL three - necked flask, add 20 mL of DMF, stir, add 0.16 g (0.001 mol) of HONB (N - hydroxy - 5 - norbornene - 2,3 - dicarboximide), add the SC - 23AA amide obtained above, add 0.25 g (0.0012 mol) of DCC, and stir and react for 2 - 3 h. Filter, pour the filtrate into pure water, precipitate solids, wash twice with water to obtain the telotristat precursor.

[0041] At 10 - 25 °C, add the telotristat precursor to a 50 mL three - necked flask, add 25 mL of trifluoroacetic acid to the reaction system, stir for 1 h. Add methyl tert - butyl ether, precipitate solids, and centrifuge to obtain the crude telotristat.

[0042] The crude telotristat is prepared by liquid phase through an ethanol / water system to obtain 2.05 g of telotristat, with a yield of 43%.

[0043] Example 3

[0044] At 10 - 25 °C, add 2.12 g (0.001 mol) of the twenty - two peptide (22 - AA) to a 50 mL two - necked flask, add 20 mL of DMF, stir, add 0.44 g (0.002 mol) of Boc anhydride, and continue to stir for 1 - 2 h. Add to pure water, precipitate solids, wash 2 times, and freeze - dry to remove water to obtain the Boc - protected twenty - two peptide (Boc - 22AA).

[0045] At 10 - 25 °C, add the obtained Boc - 22AA to a 50 mL two - necked flask, add 25 mL of DMF, add 0.184 g (0.001 mol) of HOPFP (pentafluorophenol), add 0.12 g (0.001 mol) of serine amide, add 0.25 g (0.0012 mol) of DCC, adjust the pH to 9.5 - 10.5, and continue to stir for 2 - 3 h. Filter, add the filtrate to pure water, precipitate solids, wash 2 times, and freeze - dry to obtain Boc - 23AA. Add 2N dilute hydrochloric acid, stir for 1 h, precipitate solids, filter by suction, and dry to obtain the deprotected 23AA.

[0046] At 10 - 25 °C, add 0.92 g (0.001 mol) of the side - chain active ester (OSU) to a 50 mL three - necked flask, add 20 mL of DMF, stir, add the 23AA obtained above, adjust the pH to 8.0 - 9.0, continue to react for 2 h, pour into pure water, precipitate solids, wash 2 times, and freeze - dry to obtain the 23 - AA amide with side - chain (SC - 23AA amide).

[0047] At 10 - 25 °C, add 2.6 g (0.001 mol) of the protected hexadecapeptide (16 - AA) to a 50 mL three - necked flask, add 20 mL of DMF, stir, add 0.184 g (0.001 mol) of HOPFP (pentafluorophenol), add the SC - 23AA amide obtained above, add 0.25 g (0.0012 mol) of DCC, and stir and react for 2 - 3 h. Filter, pour the filtrate into pure water, precipitate solids, wash twice with water to obtain the exenatide precursor.

[0048] At 10 - 25 °C, add the exenatide precursor to a 50 mL three - necked flask, add 25 mL of trifluoroacetic acid to the reaction system, and stir for 1 h. Add methyl tert - butyl ether, precipitate solids, and centrifuge to obtain the crude exenatide.

[0049] The crude exenatide is prepared by liquid phase through an ethanol / water system to obtain 1.86 g of exenatide with a yield of 39%.

[0050] The above embodiments are only used to illustrate the present invention and are not limitations on the present invention. Those of ordinary skill in the relevant technical field can also make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the patent protection scope of the present invention shall be defined by the claims.

Claims

1. A method for synthesizing tirzepatide, characterized in that, It includes the following consecutive steps: Step 1): Using the 22 - peptide chain shown below as a raw material, in a solvent, first obtain the Boc - protected 22 - peptide chain by biosynthesis method. After activating the Boc - protected 22 - peptide chain, react it with serine amide to obtain the Boc - protected 23 - peptide chain, and then de - Boc to obtain 23 - AA amide; Step 2): In a solvent, react the side - chain active ester shown below with the 23 - AA amide prepared in Step 1) at the ε - amino group of lysine to obtain the 23 - AA amide with a side - chain; Step 3): In a solvent, react the 23 - AA amide with a side - chain prepared in Step 2) with the protected 16 - peptide active ester shown below to obtain the telotristat precursor; Step 4): The telotristat precursor is cleaved and de - protected to obtain the crude telotristat; after purification, telotristat is obtained; The R substitution group and R1 substitution group in the protected 16 - peptide active ester and the side - chain active ester both include any one of succinimide (Su), pentafluorophenyl (PFP), phthalimide (PHT), N - 5 - norbornene - 2,3 - dicarboximide (NB); the solvent is one or more of DMF, acetonitrile, dioxane, DMSO, DMAC; in each reaction step, the pH of the reaction system is adjusted by adding a base, and the base used is one or more of triethylamine, diisopropylethylamine, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide.

2. The synthesis method of tezepelumab according to claim 1, characterized in that: In Step 1), the molar ratio of the 22 - peptide chain to (Boc)2O is 1:2; the molar ratio of the Boc - protected 22 - peptide chain to serine amide is 1:1 - 1:

2.

3. The synthesis method of teplizumab according to claim 1, wherein: In Step 2), the molar ratio of 23 - AA amide to the side - chain active ester is 1:1 - 1:1.

5.

4. The synthesis method of tirzepatide according to claim 1, wherein: In Step 3), the molar ratio of the 23 - AA amide with a side - chain to the protected 16 - peptide active ester is 1:1 - 1:1.

5.

5. The synthesis method of teplizumab according to claim 1, wherein: The mobile phase used in the purification process is alcohol and water.

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