Method for preparing side chain pentapeptide of polypeptide medicine at low temperature

Abstract

The invention provides a method for preparing Tirzeptide side chain pentapeptide based on liquid phase synthesis, which adopts ultralow-temperature refrigerated centrifugation, so that not only is a solid sample obtained, but also the purity is further improved. A solid sample is easy to transfer and can be further treated, so that the convenience of industrial production is greatly improved. The method comprises the following steps: S1, reacting mono-tert-butyl eicosanedioate with R1 to generate an intermediate I (OtBu-C20-R1); s2, reacting the intermediate I with H-Glu-OtBu to generate an intermediate II (OtBu-C20-gamma Glu (OtBu)-OH); s3, reacting the intermediate II with R1 to generate an intermediate III (OtBu-C20-gamma Glu (OtBu)-R1); s4, enabling the Boc-AEA-AEA-OH to react with the R1, so as to generate an intermediate IV (Boc-AEA-AEA-R1); s5, enabling the intermediate IV to react with Fmoc-Lys-OH and HCl to generate an intermediate V (Fmoc-Lys-(Boc-AEA-AEA)-OH); s6, removing a protecting group from the intermediate V through TFA (Trifluoroacetic Acid) to obtain an intermediate VI (Fmoc-Lys-(H-AEA-AEA)-OH); and S7, carrying out condensation on the intermediate III and the intermediate VI to obtain the Tirzepine side chain pentapeptide.

Description

Technical Field

[0001] The present invention belongs to polypeptide drugs, and particularly relates to a chemical synthesis method of tirzepatide related substances. Background Art

[0002] Tirzepatide is a GIP receptor and GLP-1 receptor agonist. A modified peptide containing 39 amino acids with a C20 fatty acid group, which can bind to albumin and extend the half-life to 5 days, so it can be injected once a week. Tirzepatide enhances the first-phase and second-phase insulin secretion in a glucose-dependent manner and reduces the glucagon level.

[0003] Glucagon-like peptide-1 (GLP-1) plays an important role in the field of diabetes treatment. It can not only control blood sugar and reduce weight, but also has a cardiovascular protective effect. In the previous exploration stage, another target, glucose-dependent insulinotropic polypeptide (GIP), was considered to have no potential for hypoglycemic treatment. However, new evidence shows that compared with the use of the two hormones alone, GLP-1 + GIP shows a synergistic effect, that is, 1 + 1 > 2, resulting in a significant increase in insulin response and glucagon inhibition response. Tirzepatide is a novel GIP / GLP-1 receptor dual agonist with potent blood sugar control and weight loss effects. It was approved by the US FDA in May 2022 and is used as an adjunct to diet and exercise to improve blood sugar control in adult patients with type 2 diabetes. In addition, it can also be found in clinical trials that it is also conducting clinical trials on metabolic disorder-related indications such as sleep apnea syndrome, heart failure, and non-alcoholic steatohepatitis (NASH). Summary of the Invention

[0004] The purpose of the present invention is to provide a synthesis method of the side-chain pentapeptide, an important intermediate in the chemical synthesis of tirzepatide.

[0005] The present invention provides a method for preparing the Tirzepatide side-chain pentapeptide based on liquid-phase synthesis. The synthesis technology of the present invention can be combined with ultra-low temperature freezing centrifugation, which not only obtains solid samples, but also further improves the purity. The solid samples are easy to transfer and can also be further processed, greatly increasing the convenience of industrial production. The method of the present invention includes:

[0006] S1. Monotert-butyl eicosanedioate reacts with R1 to generate intermediate one (OtBu-C20-R1);

[0007] S2. Intermediate one reacts with H-Glu-OtBu to generate intermediate two (OtBu-C20-γGlu(OtBu)-OH);

[0008] Intermediate S3 reacts with R1 to form Intermediate S3 (OtBu-C20-γGlu(OtBu)-R1);

[0009] S4, Boc-AEEA-AEEA-OH reacts with R1 to form Intermediate S4 (Boc-AEEA-AEEA-R1);

[0010] Intermediate S4 reacts with Fmoc-Lys-OH.HCl to form Intermediate S5 (Fmoc-Lys-(Boc-AEEA-AEEA)-OH);

[0011] S6, Intermediate S5 is deprotected with TFA to obtain Intermediate S6 (Fmoc-Lys-(H-AEEA-AEEA)-OH);

[0012] S7, Intermediate S3 is condensed with Intermediate S6 to obtain the five-peptide side chain of Tirzepatide.

[0013] Among them, R1 can be selected from HOSU, HOBt, HOAt, pentafluorophenol, p-nitrophenol, etc.

[0014] The reaction equation is:

[0015]

[0016] According to one aspect of the present invention, a method for preparing Intermediate S1,

[0017]

[0018] comprises the following steps:

[0019] OtBu-C20 and R1 are reacted with a suitable condensing agent to form Intermediate S1, and Intermediate S1 with high purity is obtained by recrystallization.

[0020] When R1 is HOSU, more preferably pentafluorophenol, the condensing agent is DCC, more preferably EDC.HCl, and the solvent is one or a mixed solution of common low-boiling-point acetonitrile, ethyl acetate, DCM, and tetrahydrofuran, more preferably acetonitrile.

[0021] The reaction temperature is -5°C to 5°C;

[0022] The reaction time is 10 to 12 hours;

[0023] The post-treatment is carried out by pickling, alkali washing, water washing, concentrating the organic phase, co-evaporating with isopropanol, and adding IPE for crystallization.

[0024] According to one aspect of the present invention, a method for preparing Intermediate S2,

[0025]

[0026] It includes the following steps:

[0027] React intermediate one and H-Glu-OtBu in a suitable solvent under the catalysis of a suitable inorganic base to generate intermediate two, and obtain high-purity intermediate two through recrystallization.

[0028] The reaction solvent is tetrahydrofuran / water or acetonitrile / water, preferably tetrahydrofuran / water;

[0029] The inorganic base is NaHCO3 or Na2CO3, preferably Na2CO 3；

[0030] The post-treatment adopts pickling, alkali washing, and water washing, and the recrystallization adopts IPE;

[0031] According to one aspect of the present invention, the preparation method of intermediate three

[0032]

[0033] It includes the following steps:

[0034] R1 adopts HOSU, more preferably pentafluorophenol, the condensing agent adopts DCC, more preferably EDC.HC, the solvent adopts one or a mixed solution of commonly used low-boiling-point acetonitrile, ethyl acetate, DCM, and tetrahydrofuran, and more preferably DCM.

[0035] The reaction temperature is -5°C to 5°C;

[0036] The reaction time is 10 to 12 h;

[0037] The post-treatment adopts pickling, alkali washing, and water washing, and the recrystallization adopts n-Hex.

[0038] According to one aspect of the present invention, the preparation method of intermediate four,

[0039]

[0040] It includes the following steps:

[0041] React Boc-AEEA-AEEA-OH and R1 in a suitable condensing agent to generate intermediate four, and further perform pickling and alkali washing to improve the sample purity.

[0042] R1 adopts HOSU, more preferably pentafluorophenol, the condensing agent adopts DCC, more preferably EDC.HC, the solvent adopts one or a mixed solution of commonly used low-boiling-point acetonitrile, ethyl acetate, DCM, and tetrahydrofuran, and more preferably DCM.

[0043] The reaction temperature is -5°C to 5°C;

[0044] The reaction time is 10 - 12 h

[0045] The post-treatment adopts pickling, alkali washing and water washing.

[0046] According to one aspect of the present invention, a method for preparing intermediate five

[0047]

[0048] comprises the following steps:

[0049] React intermediate four and Fmoc-Lys-OH·HCl in a suitable solvent under the catalysis of a suitable inorganic base to form intermediate five, and perform low-temperature centrifugation and recrystallization treatment to improve the sample purity.

[0050] The reaction solvent is tetrahydrofuran / water or acetonitrile / water, preferably tetrahydrofuran / water;

[0051] The inorganic base is NaHCO3 or Na2CO3, preferably Na2CO3;

[0052] The post-treatment adopts pickling, alkali washing and water washing, and ethyl acetate is used for ultra-low temperature centrifugation and recrystallization.

[0053] According to one aspect of the present invention, a method for preparing intermediate six

[0054]

[0055] comprises the following steps:

[0056] The cleavage acid is one of TFA, hydrochloric acid, sulfuric acid, trifluoromethanesulfonic acid, preferably TFA;

[0057] The cleavage adopts a mixed reagent, TFA / DCM;

[0058] The cleavage time is 2 - 3 h;

[0059] The cleavage temperature is 20 - 25 °C;

[0060] The post-treatment adopts IPE sedimentation.

[0061] According to one aspect of the present invention, a method for preparing the side-chain pentapeptide of the present invention

[0062]

[0063] comprises the following steps:

[0064] React intermediate three and intermediate six in a suitable solvent under the catalysis of a suitable inorganic base to form the side-chain pentapeptide, and perform low-temperature centrifugation and recrystallization treatment to obtain a high-purity sample.

[0065] The reaction solvent is tetrahydrofuran / water or acetonitrile / water, preferably tetrahydrofuran / water;

[0066] The inorganic base is NaHCO3 or Na2CO3, preferably Na2CO3;

[0067] The post-treatment adopts pickling, alkali washing, and water washing. Acetonitrile is used for low-temperature centrifugal recrystallization. The temperature is controlled at -20°C and left to stand. An oily substance first precipitates at the bottom layer and then turns into crystals, which are not easy to take out and difficult to filter. After centrifugation by an ultra-low temperature freezing centrifuge, the layering is good, presenting a whole block, easy to take out and weigh, with less loss. Description of the Drawings

[0068] Figure 1 The detection spectrum of Intermediate 1 in the embodiment of the present invention;

[0069] Figure 2 The detection spectrum of Intermediate 2 in the embodiment of the present invention;

[0070] Figure 3 The detection spectrum of Intermediate 3 in the embodiment of the present invention;

[0071] Figure 4 The detection spectrum of Intermediate 4 in the embodiment of the present invention;

[0072] Figure 5 The detection spectrum of Intermediate 5 in the embodiment of the present invention;

[0073] Figure 6 The detection spectrum of Intermediate 6 in the embodiment of the present invention;

[0074] Figure 7 The detection spectrum of the side-chain pentapeptide generated by the reaction in the embodiment of the present invention. Detailed Description of the Invention

[0075] Example 1

[0076] OtBu-C20 (119.58 g, 300 mmol) and pentafluorophenol (60.74 g, 330 mmol, 1.1 eq) were added to a reaction kettle, 3000 ml of acetonitrile was added and stirred to dissolve. The temperature was controlled at -5°C to 5°C, and a solution of EDC.HCl (63.26 g, 330 mmol, 1.1 eq) in acetonitrile (500 ml) was dropped in. After the dropping was completed, the reaction was carried out at -5°C to 5°C for 10 - 12 h. After the reaction was completed, it was pickled, alkali-washed, and water-washed, the organic phase was concentrated, co-evaporated with isopropanol, and IPE was added for crystallization to obtain 159.26 g of Intermediate 1. The detection results are as Figure 1 , with a yield of 94.01% and a purity of 98.76%.

[0077] Example 2

[0078] Add intermediate I (112.93 g, 200 mmol) and H-Glu-OtBu (44.70 g, 220 mmol, 1.1 eq) to the reaction kettle, add 2200 ml of THF, stir to dissolve, control the temperature at 20 °C to 25 °C, and dropwise add an aqueous solution of Na2CO3 (31.80 g, 300 mmol, 1.5 eq) in purified water (500 ml). React at 20 °C to 25 °C for 8 - 10 h. After the reaction is completed, wash with acid, wash with alkali, and wash with water. Rotate and evaporate to concentrate the organic phase, and recrystallize with IPE to obtain 107.42 g of intermediate II. The test results are as Figure 2 , with a yield of 92% and a purity of 98.39%.

[0079] Example 3

[0080] Add intermediate II (87.57 g, 150 mmol) and pentafluorophenol (30.37 g, 165 mmol, 1.1 eq) to the reaction kettle, add 2000 ml of DCM, stir to dissolve, control the temperature at -5 °C to 5 °C, and dropwise add a solution of EDC.HCl (31.63 g, 165 mmol, 1.1 eq) in DCM (500 ml). After the addition is complete, react at -5 °C to 5 °C for 10 - 12 h. After the reaction is completed, wash with acid, wash with alkali, and wash with water. Rotate and evaporate to concentrate the organic phase, and recrystallize with n-Hex to obtain 106.88 g of intermediate III. The test results are as Figure 3 , with a yield of 95.02% and a purity of 98.88%.

[0081] Example 4

[0082] Add Boc-AEEA-AEEA-OH (122.52 g, 300 mmol) and pentafluorophenol (60.74 g, 330 mmol, 1.1 eq) to the reaction kettle, add 3000 ml of DCM, control the temperature at -5 °C to 5 °C, and dropwise add a solution of EDC.HCl (63.26 g, 330 mmol, 1.1 eq) in DCM (500 ml). After the addition is complete, react at -5 °C to 5 °C for 10 - 12 h. After the reaction is completed, wash with acid, wash with alkali, and wash with water. Concentrate the organic phase to obtain 167.17 g of intermediate IV. The test results are as Figure 4 , with a yield of 97% and a purity of 99.31%.

[0083] Example 5

[0084] The intermediate four (126.38 g, 220 mmol) and Fmoc-Lys-OH·HCl (97.98 g, 242 mmol, 1.1 eq) were added to a reaction kettle, 2500 ml of THF was added, and the mixture was stirred until dissolved. The temperature was controlled at 20°C - 25°C, and an aqueous solution of Na2CO3 (34.98 g, 330 mmol, 1.5 eq) in purified water (600 ml) was added dropwise. The reaction was carried out at 20°C - 25°C for 8 - 10 h. After the reaction, it was washed with acid, washed with alkali, and washed with water. The organic phase was concentrated by rotary evaporation, dissolved in ethyl acetate, and frozen and centrifuged at -20°C to obtain 148.59 g of intermediate five. The test results are as Figure 5 , with a yield of 89% and a purity of 98.83%.

[0085] Example 6

[0086] Intermediate five (136.6 g, 180 mmol) was added and dissolved in 270 ml of DCM, and then added dropwise to a mixed solution of 270 ml of DCM and 540 ml of TFA. The reaction was carried out at 20°C - 25°C for 3 h. After the reaction, the reaction solution was added to 4000 ml of IPE stirred at -10°C, and stirring was maintained for 20 min. Then it was filtered to obtain 113.82 g of intermediate six. The test results are as Figure 6 , with a yield of 95.99% and a purity of 98.69%.

[0087] Example 7

[0088] The intermediate three (81.7 g, 120 mmol) and intermediate six (83 g, 126 mmol, 1.05 eq) were added to a reaction kettle, 1600 ml of THF was added, and the mixture was stirred until dissolved. The temperature was controlled at 20°C - 25°C, and an aqueous solution of Na2CO3 (19.08 g, 180 mmol, 1.5 eq) in purified water (400 ml) was added dropwise. The reaction was carried out at 20°C - 25°C for 8 - 10 h. After the reaction, it was washed with acid, washed with alkali, and washed with water. The organic phase was concentrated by rotary evaporation, dissolved in acetonitrile, and frozen and centrifuged at -20°C to obtain 133.73 g of the Tirzepatide side-chain pentapeptide. The test results are as Figure 7 , with a yield of 91% and a purity of 99.77%.

[0089] It should be noted that the above preferred embodiments are only used to illustrate the technical concept and characteristics of the present invention. The purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and it cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing a side-chain pentapeptide of a polypeptide drug at low temperature, characterized in that, The polypeptide drug is tirzepatide. The method for preparing the side-chain pentapeptide of the polypeptide drug at low temperature includes the preparation of intermediate I, and the steps are as follows: React OtBu-C20 and R1 with a suitable condensing agent to generate intermediate I, and obtain high-purity intermediate I through recrystallization; R1 uses HOSU or pentafluorophenol, the condensing agent uses DCC or EDC·HCl, and the solvent uses one or a mixed solution of acetonitrile, ethyl acetate, DCM, and tetrahydrofuran; The reaction temperature is -5°C to 5°C; The reaction time is 10 to 12 h; The post-treatment uses acid washing, alkali washing, and water washing, concentrates the organic phase, co-evaporates with isopropanol, and adds IPE for crystallization.

2. The method for preparing the side-chain pentapeptide of the polypeptide drug at low temperature according to claim 1, wherein, The method for preparing the side-chain pentapeptide of the polypeptide drug at low temperature includes the preparation of intermediate II, and the steps are as follows: React the intermediate I and H-Glu-OtBu in a solvent under the catalysis of an inorganic base to generate intermediate II, and obtain high-purity intermediate II through recrystallization; The reaction solvent uses tetrahydrofuran / water or acetonitrile / water; The inorganic base uses NaHCO3 or Na2CO3; The post-treatment uses acid washing, alkali washing, and water washing, and the recrystallization uses IPE.

3. The method for preparing the side-chain pentapeptide of the polypeptide drug at low temperature according to claim 2, wherein, The method for preparing the side-chain pentapeptide of the polypeptide drug at low temperature includes the preparation of intermediate III, and the method is as follows: R1 uses HOSU or pentafluorophenol, the condensing agent uses DCC or EDC·HCl, and the solvent uses one or a mixed solution of acetonitrile, ethyl acetate, DCM, and tetrahydrofuran; The reaction temperature is -5°C to 5°C; The reaction time is 10 to 12 h; The post-treatment uses acid washing, alkali washing, and water washing, and the recrystallization uses n-Hex.

4. The method for preparing the side-chain pentapeptide of the polypeptide drug at low temperature according to claim 3, wherein The method for preparing the side-chain pentapeptide of the polypeptide drug at low temperature includes the preparation of intermediate IV, and the steps are as follows: React Boc-AEEA-AEEA-OH and R1 in a suitable condensing agent to generate intermediate IV, and further perform acid washing and alkali washing to improve the sample purity; R1 uses HOSU or pentafluorophenol, the condensing agent uses DCC or EDC·HCl, and the solvent uses one or a mixed solution of acetonitrile, ethyl acetate, DCM, and tetrahydrofuran; The reaction temperature is -5°C to 5°C; The reaction time is 10 - 12 h; The post-treatment uses acid washing, alkali washing, and water washing.

5. The method for preparing the side-chain pentapeptide of the polypeptide drug at low temperature according to claim 4, wherein, The method for preparing the side-chain pentapeptide of the polypeptide drug at low temperature includes the preparation of intermediate V, and the steps are as follows: React intermediate IV and Fmoc-Lys-OH·HCl in a solvent under the catalysis of an inorganic base to generate intermediate V, and perform low-temperature centrifugation and recrystallization treatment to improve the sample purity; The reaction solvent uses tetrahydrofuran / water or acetonitrile / water; The inorganic base uses NaHCO3 or Na2CO3; The post-treatment uses acid washing, alkali washing, and water washing, and the ultra-low temperature centrifugation and recrystallization use ethyl acetate.

6. The method for preparing the side-chain pentapeptide of the polypeptide drug at low temperature according to claim 5, wherein The method for preparing the side-chain pentapeptide of the polypeptide drug at low temperature includes the preparation of intermediate VI, and the steps are as follows: The cleavage acid uses one of TFA, hydrochloric acid, sulfuric acid, and trifluoromethanesulfonic acid; The cleavage uses a mixed reagent, TFA / DCM; The cleavage time is 2 to 3 h; The cleavage temperature is 20 to 25°C; The post-treatment uses IPE sedimentation.

7. The method for preparing the side-chain pentapeptide of the polypeptide drug at low temperature according to claim 6, wherein The method for preparing the side-chain pentapeptide of the polypeptide drug at low temperature further includes the following steps: React the intermediate three and the intermediate six in a solvent under the catalysis of an inorganic base to generate the telotristat side-chain pentapeptide, and obtain a high-purity sample through low-temperature centrifugation and recrystallization treatment; The reaction solvent is tetrahydrofuran / water or acetonitrile / water; The inorganic base is NaHCO3 or Na2CO3.

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