Method for preparing side chain intermediates of telpotitide
By using the method of forming amide bonds in one pot when preparing the terpopeptide side chain intermediate, the intermediate purification process is simplified, the experimental steps are optimized, the cost is reduced, and the problems of complex and high cost of intermediate purification in the prior art are solved, thereby achieving efficient and economical preparation of terpopeptide intermediates.
Patent Information
- Application Number
- CN202411959267.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-30
- Publication Date
- 2025-04-04
AI Technical Summary
In the prior art, when preparing terpopeptide side chain intermediates, the intermediate purification process is complex, resulting in a variety of experimental steps and high costs, which makes it difficult to meet market demand.
The method of forming amide bonds in one pot is adopted to reduce the intermediate purification process and optimize the experimental steps. By reacting 20-(tert-butoxy)-20-oxoeicosanoic acid with alkyl chloroformate, combining sodium hydroxide solution, adjusting the acid and purifying the product, and purifying it through ethyl acetate extraction and recrystallization to obtain a terpopeptide side chain intermediate.
The intermediate purification process is simplified, the experimental steps are optimized, the experimental costs are reduced, the production efficiency is improved, and the quality and efficacy of terpopeptide are ensured.
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Figure CN119751308A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a method for preparing a telpotide side chain intermediate. Background Art
[0002] Tirzepatide is a dual agonist of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors. It integrates the effects of two insulinotropic peptides into a single molecule and activates the hormone receptors involved in blood sugar control-GIP and GLP-1 two incretin receptors through a dual mechanism of action. It is administered by subcutaneous injection once a week to achieve the goal of controlling blood sugar. In May 2023, Tirzepatide was approved for marketing by the US FDA as a "first-in-class" drug. It is the first new generation of diabetes drug launched in the past decade. The molecular weight of tirzepatide is 4813.53 and the molecular formula is C 225 H 348 N 48 O 68 , is a modified peptide based on the natural GIP sequence consisting of 9 amino acids. It contains two non-coding amino acids (aminoisobutyric acid, Aib) at the 2nd and 13th positions, and a C-terminal amide is connected to 1,20-eicosanedioic acid through a hydrophilic linker of the 20th lysine residue, so that it can bind to albumin, prolong the half-life, and can be administered subcutaneously once a week. Telpotide can activate the receptors of two natural incretin hormones, GIP and GLP-1, in the human body. The two mechanisms of action are synergistic and complementary, achieving a "1+1>2" cohesive effect in many aspects such as blood sugar control and weight loss, and ultimately further improving the safety and efficacy of the drug. Studies have shown that telpotide has achieved good weight loss effects in obese patients with and without diabetes.
[0003] The structural formula of Tirzepatide side chain intermediate (CAS: 2460751-66-8) is as follows:
[0004] Fmoc-L-Lys[C20-OtBu-Glu(OtBu)-AEEA-AEEA]-OH is a key intermediate in the synthesis of Tirzepatide.
[0005] The synthesis of side chain intermediates is one of the key steps in the production of telpotide. By synthesizing intermediates, a stable supply of raw materials for telpotide can be ensured to meet market demand. The synthesis process of intermediates can be strictly quality controlled to ensure that the quality of the final product meets the standards. This helps to improve the efficacy and safety of the drug. By optimizing the synthesis process of intermediates, production efficiency can be improved and production costs can be reduced. This helps to reduce the price of the drug so that more patients can benefit from the treatment of the drug. Research and development on the synthesis of intermediates can provide new ideas and methods for drug research and development. This helps to promote drug innovation and provide more options for the treatment of other diseases. Summary of the invention
[0006] The invention provides a method for preparing a telpotide side chain intermediate. The method forms an amide bond in one pot, reduces the intermediate purification process, optimizes the experimental steps, and reduces the experimental cost.
[0007] The technical solution of the present invention is as follows:
[0008] The present invention provides a method for preparing a telpotide side chain intermediate, comprising the following steps:
[0009] (1) 20-(tert-butoxy)-20-oxoeicosanoic acid and N,N-diisopropylethylamine are dissolved in an organic solvent I, and then alkyl chloroformate is added, followed by reaction at room temperature to generate M1;
[0010] (2) After step (1), a strong base solution containing SM2 is added, and after the reaction is complete, the acid is adjusted, the liquids are separated, ethyl acetate is added to the aqueous phase for extraction, the organic phases are combined, concentrated under reduced pressure, and then recrystallized and purified with water and tetrahydrofuran, and dried to obtain M2 solid;
[0011] (3) M2 and N,N-diisopropylethylamine are dissolved in an organic solvent II, and then alkyl chloroformate is added, followed by reaction at room temperature to generate M3;
[0012] (4) After step (3), a strong base solution containing SM3 is added, and after the reaction is complete, the acid is adjusted, the liquids are separated, ethyl acetate is added to the aqueous phase for extraction, the organic phases are combined, concentrated under reduced pressure, and then purified by recrystallization with water and tetrahydrofuran, and dried to obtain M4 solid;
[0013] (5) M4 and N,N-diisopropylethylamine are dissolved in an organic solvent III, and then alkyl chloroformate is added, followed by reaction at room temperature to generate M5;
[0014] (6) After step (5), a strong base solution containing SM4 is added, and after the reaction is complete, the acid is adjusted, the liquids are separated, ethyl acetate is added to the aqueous phase for extraction, the organic phases are combined, concentrated under reduced pressure, purified by recrystallization with water, and dried to obtain M6 solid;
[0015] Wherein, SM2 is 1-tert-butyl L-glutamic acid;
[0016] SM3 is 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid;
[0017] SM4 is Fmoc-lysine;
[0018] M6 is a telpotide side chain intermediate, and its structural formula is shown in formula (I):
[0019]
[0020] Reaction equation of the present invention is as follows:
[0021]
[0022] In a specific embodiment, in step (1), the equivalent of N,N-diisopropylethylamine added is 1.2-3; the organic solvent I is at least one of dichloromethane, N,N-dimethylformamide, ethanol, and acetonitrile; the volume of the organic solvent I added is 9-12 times the mass of the substrate; preferably, the volume of the organic solvent I added is 10 times the mass of the substrate; the alkyl chloroformate is at least one of ethyl chloroformate, propyl chloroformate, isopropyl chloroformate, isobutyl chloroformate, and n-butyl chloroformate; the equivalent of the alkyl chloroformate added is 1.1-1.4; preferably, the equivalent of the alkyl chloroformate added is 1.2.
[0023] In a specific embodiment, in step (2), the equivalent of SM2 added is 1.1-1.4; preferably, the equivalent of SM2 added is 1.2; the strong alkaline solution of SM2 is a sodium hydroxide solution of SM2 or a potassium hydroxide solution of SM2; in the strong alkaline solution of SM2, the dosage ratio of SM2, strong alkali and water is 1.12g:0.22-0.36g:2-10ml; the pH after acid adjustment is 2-4; the volume ratio of water and tetrahydrofuran added during recrystallization is 4-5:0-1; preferably, the volume ratio of water and tetrahydrofuran added during recrystallization is 4:1.
[0024] In a specific embodiment, in step (3), the equivalent of N,N-diisopropylethylamine added is 1.2-3; the organic solvent II is at least one of dichloromethane, N,N-dimethylformamide, ethanol, and acetonitrile; the volume of organic solvent II added is 9-12 times the mass of the substrate; preferably, the volume of organic solvent II added is 10 times the mass of the substrate; the alkyl chloroformate is at least one of ethyl chloroformate, propyl chloroformate, isopropyl chloroformate, isobutyl chloroformate, and n-butyl chloroformate; the equivalent of the alkyl chloroformate added is 1.1-1.4; preferably, the equivalent of the alkyl chloroformate added is 1.2.
[0025] In a specific embodiment, in step (4), the equivalent of SM3 added is 1.1-1.4; preferably, the equivalent of SM3 added is 1.3; the strong alkaline solution of SM3 is a sodium hydroxide solution of SM3 or a potassium hydroxide solution of SM3; in the strong alkaline solution of SM3, the dosage ratio of SM3, strong alkali and water is 1.38g:0.17-0.51g:2-10ml; the pH after acid adjustment is 2-4; the volume ratio of water and tetrahydrofuran added during recrystallization is 4-5:0-1; preferably, the volume ratio of water and tetrahydrofuran added during recrystallization is 4:1.
[0026] In a specific embodiment, in step (5), the equivalent of N,N-diisopropylethylamine added is 1.2-3; the organic solvent III is at least one of dichloromethane, N,N-dimethylformamide, ethanol, and acetonitrile; the volume of the organic solvent III added is 9-12 times the mass of the substrate; preferably, the volume of the organic solvent III added is 10 times the mass of the substrate; the alkyl chloroformate is at least one of ethyl chloroformate, propyl chloroformate, isopropyl chloroformate, isobutyl chloroformate, and n-butyl chloroformate; the equivalent of the alkyl chloroformate added is 1.1-1.4; preferably, the equivalent of the alkyl chloroformate added is 1.2.
[0027] In a specific embodiment, in step (6), the equivalent of SM4 added is 1.1-1.4; preferably, the equivalent of SM4 added is 1.3; the strong alkaline solution of SM4 is a sodium hydroxide solution of SM4 or a potassium hydroxide solution of SM4; in the strong alkaline solution of SM4, the dosage ratio of SM4, strong base and water is 1.10g:0.17-0.51g:2-10ml; the pH after acid adjustment is 2-4.
[0028] In one embodiment, the reactions in steps (1) to (6) are all tracked by TCL.
[0029] Compared with the prior art, the present invention has the following beneficial effects:
[0030] The present invention uses 20-(tert-butoxy)-20-oxoeicosanoic acid as a starting material, uses alkyl chloroformate, forms an active anhydride intermediate, then adds a sodium hydroxide solution of another substrate, reacts to form a sodium salt of the product, adjusts the acid, forms the product, extracts the product with ethyl acetate, combines the organic phases, concentrates, and then purifies. The synthetic method forms an amide bond in one pot, reduces the intermediate purification process, optimizes the experimental steps, and reduces the experimental cost. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 The structure diagram of M6 in Example 1 is determined by hydrogen nuclear magnetic resonance spectrum;
[0032] Figure 2This is the mass spectrometry analysis result of M6 in Example 1;
[0033] Figure 3 This is the HPLC detection spectrum of M6 in Example 1. DETAILED DESCRIPTION
[0034] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.
[0035] Example 1
[0036] (1) Put 2 g (5.016 mmol) of 20-(tert-butoxy)-20-oxoeicosanoic acid into a reaction container, add 1.70 g (13.14 mmol) of N,N-diisopropylethylamine, and then add 20 mL of dichloromethane to dissolve. After the solution is clear, add 0.74 g (6.019 mmol) of isopropyl chloroformate and stir the reaction at room temperature. Follow the reaction by TLC until the reaction of 20-(tert-butoxy)-20-oxoeicosanoic acid is complete. Then prepare SM2 (1-tert-butyl L-glutamic acid) sodium hydroxide solution: dissolve 1.12 g SM2 (6.019 mmol) and 0.24 g (6.020 mmol) of sodium hydroxide in 10 mL of pure water. A sodium hydroxide solution of SM2 was added to the reaction vessel. The reaction was monitored by TLC. After the intermediate was completely reacted, hydrochloric acid was added to adjust the pH to about 3-4. The liquids were separated. 40 ml of ethyl acetate was added to the aqueous phase for extraction. The extraction was repeated 3 times. TLC was monitored until there was no M2. The organic phases were combined and concentrated under reduced pressure at 50°C. After concentration, 16 ml of water and 4 ml of tetrahydrofuran were added. The mixture was heated to reflux at 60°C, cooled for crystallization, filtered, and dried under reduced pressure at 50°C to obtain 2.52 g of white solid M2 with a yield of 86.0%.
[0037] (2) 2 g of M2 (3.426 mmol) was placed in a reaction vessel, 1.16 g of N,N-diisopropylethylamine (7.743 mmol) was added, and 20 mL of dichloromethane was added to dissolve. After the solution was clear, 0.50 g of isopropyl chloroformate (4.111 mmol) was added and stirred at room temperature for reaction. The reaction was monitored by TLC until the reaction of M2 was complete. Then, SM3 (AEEA-AEEA) sodium hydroxide solution was prepared: 1.38 g of SM3 (4.454 mmol) and 0.17 g of sodium hydroxide (4.111 mmol) were dissolved in 10 mL of pure water. SM3 sodium hydroxide solution was added to the reaction vessel. The reaction was monitored by TLC until the reaction of the intermediate was complete. Hydrochloric acid was added to adjust the pH to about 3-4, and the liquids were separated. 40 ml of ethyl acetate was added to the aqueous phase for extraction. The reaction was repeated 3 times. TLC was monitored until there was no M4 in the aqueous phase. The organic phases were combined and concentrated under reduced pressure at 50°C. After concentration, 16 mL of water and 4 mL of tetrahydrofuran were added, and the mixture was heated to reflux at 60°C. The mixture was cooled to crystallize, filtered, and dried under reduced pressure at 50°C to obtain 2.30 g of a white solid M4 with a yield of 88.1%.
[0038] (3) 2 g of M4 (2.288 mmol) was placed in a reaction vessel, and 0.77 g of N,N-diisopropylethylamine (5.995 mmol) was added. Then, 20 mL of dichloromethane was added to dissolve the mixture. After the mixture was dissolved, 0.34 g of isopropyl chloroformate (2.746 mmol) was added and the mixture was stirred at room temperature for reaction. The reaction was monitored by TLC until the reaction of M4 was complete. Then, a sodium hydroxide solution of SM4 (Fmoc-lysine) was prepared: 1.10 g of SM4 (2.975 mmol) and 0.17 g of sodium hydroxide (2.746 mmol) were dissolved in 10 mL of pure water. The sodium hydroxide solution of SM4 was added to the reaction vessel. The reaction was monitored by TLC until the reaction of the intermediate was complete. Then, hydrochloric acid was added to adjust the pH to about 3-4. The mixture was separated. 40 mL of ethyl acetate was added to the aqueous phase for extraction. The reaction was repeated three times. TLC was monitored until there was no M6 in the aqueous phase. The organic phases were combined and concentrated under reduced pressure at 50°C. After concentration, 20 mL of water was added, and the mixture was heated to reflux at 60°C. The mixture was cooled to crystallize, filtered, and dried under reduced pressure at 50°C to obtain 2.53 g of a white solid M6 with a yield of 90.3%.
[0039] The structure of M6 determined by H NMR spectrum is shown in Figure 1 shown.
[0040] The mass spectrometry results of M6 are shown in Figure 2 As shown, the relative molecular mass is 1223.
[0041] Purity identification of M6: M6 was tested by HPLC. The conditions of HPLC were as follows:
[0042] Mobile phase: A 0.1% trifluoroacetic acid acetonitrile B 0.1% trifluoroacetic acid water
[0043] Flow rate: 1mL / min
[0044] Detection wavelength: 210nm, 254nm
[0045] Column temperature: 40°C
[0046] Chromatographic column: phenomenex gemini 5u C18110A 150*4.60mm 5micron
[0047] Elution procedure:
[0048] Time (min) A(％) B(％) 0 28 72 20 100 0 40 100 0 41 28 72 50 28 72
[0049] M6 was detected by HPLC. Figure 3 As shown, the retention time of M6 is 15.776 min and the purity is 99.15%.
[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for preparing a telpotide side chain intermediate, characterized in that: The steps include: (1) 20-(tert-butoxy)-20-oxoeicosanoic acid and N,N-diisopropylethylamine are dissolved in an organic solvent I, and then alkyl chloroformate is added, followed by reaction at room temperature to generate M1; (2) After step (1), a strong base solution containing SM2 is added, and after the reaction is complete, the acid is adjusted, the liquids are separated, ethyl acetate is added to the aqueous phase for extraction, the organic phases are combined, concentrated under reduced pressure, and then recrystallized and purified with water and tetrahydrofuran, and dried to obtain M2 solid; (3) M2 and N,N-diisopropylethylamine are dissolved in an organic solvent II, and then alkyl chloroformate is added, followed by reaction at room temperature to generate M3; (4) After step (3), a strong base solution containing SM3 is added, and after the reaction is complete, the acid is adjusted, the liquids are separated, ethyl acetate is added to the aqueous phase for extraction, the organic phases are combined, concentrated under reduced pressure, and then purified by recrystallization with water and tetrahydrofuran, and dried to obtain M4 solid; (5) M4 and N,N-diisopropylethylamine are dissolved in an organic solvent III, and then alkyl chloroformate is added, followed by reaction at room temperature to generate M5; (6) After step (5), a strong base solution containing SM4 is added, and after the reaction is complete, the acid is adjusted, the liquids are separated, ethyl acetate is added to the aqueous phase for extraction, the organic phases are combined, concentrated under reduced pressure, purified by recrystallization with water, and dried to obtain M6 solid; Wherein, SM2 is 1-tert-butyl L-glutamic acid; SM3 is 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid; SM4 is Fmoc-lysine; M6 is a telpotide side chain intermediate, and its structural formula is shown in formula (I):
2. The method for preparing a telpotide side chain intermediate according to claim 1, characterized in that: In step (1), the equivalent of N, N-diisopropylethylamine added is 1.2-3; the organic solvent I is at least one of dichloromethane, N, N-dimethylformamide, ethanol, and acetonitrile; the volume of the organic solvent I added is 9-12 times the mass of the substrate; preferably, the volume of the organic solvent I added is 10 times the mass of the substrate; the alkyl chloroformate is at least one of ethyl chloroformate, propyl chloroformate, isopropyl chloroformate, isobutyl chloroformate, and n-butyl chloroformate; the equivalent of the alkyl chloroformate added is 1.1-1.4; preferably, the equivalent of the alkyl chloroformate added is 1.
2.
3. The method for preparing a telpotide side chain intermediate according to claim 1, characterized in that: In step (2), the equivalent of SM2 added is 1.1-1.4; preferably, the equivalent of SM2 added is 1.2; the strong alkaline solution of SM2 is a sodium hydroxide solution of SM2 or a potassium hydroxide solution of SM2; in the strong alkaline solution of SM2, the dosage ratio of SM2, strong alkali and water is 1.12g:0.22-0.36g:2-10ml; the pH after acid adjustment is 2-4; the volume ratio of water and tetrahydrofuran added during recrystallization is 4-5:0-1; preferably, the volume ratio of water and tetrahydrofuran added during recrystallization is 4:
1.
4. The method for preparing a telpotide side chain intermediate according to claim 1, characterized in that: In step (3), the equivalent of N,N-diisopropylethylamine added is 1.2-3; the organic solvent II is at least one of dichloromethane, N,N-dimethylformamide, ethanol, and acetonitrile; the volume of organic solvent II added is 9-12 times the mass of the substrate; preferably, the volume of organic solvent II added is 10 times the mass of the substrate; the alkyl chloroformate is at least one of ethyl chloroformate, propyl chloroformate, isopropyl chloroformate, isobutyl chloroformate, and n-butyl chloroformate; the equivalent of the alkyl chloroformate added is 1.1-1.4; preferably, the equivalent of the alkyl chloroformate added is 1.
2.
5. The method for preparing a telpotide side chain intermediate according to claim 1, characterized in that: In step (4), the equivalent of SM3 added is 1.1-1.4; preferably, the equivalent of SM3 added is 1.3; the strong alkaline solution of SM3 is a sodium hydroxide solution of SM3 or a potassium hydroxide solution of SM3; in the strong alkaline solution of SM3, the dosage ratio of SM3, strong base and water is 1.38g:0.17-0.51g:2-10ml; the pH after acid adjustment is 2-4; the volume ratio of water and tetrahydrofuran added during recrystallization is 4-5:0-1; preferably, the volume ratio of water and tetrahydrofuran added during recrystallization is 4:
1.
6. The method for preparing a telpotide side chain intermediate according to claim 1, characterized in that: In step (5), the equivalent of N,N-diisopropylethylamine added is 1.2-3; the organic solvent III is at least one of dichloromethane, N,N-dimethylformamide, ethanol, and acetonitrile; the volume of the organic solvent III added is 9-12 times the mass of the substrate; preferably, the volume of the organic solvent III added is 10 times the mass of the substrate; the alkyl chloroformate is at least one of ethyl chloroformate, propyl chloroformate, isopropyl chloroformate, isobutyl chloroformate, and n-butyl chloroformate; the equivalent of the alkyl chloroformate added is 1.1-1.4; preferably, the equivalent of the alkyl chloroformate added is 1.
2.
7. The method for preparing a telpotide side chain intermediate according to claim 1, characterized in that: In step (6), the equivalent of SM4 added is 1.1-1.4; preferably, the equivalent of SM4 added is 1.3; the strong alkaline solution of SM4 is a sodium hydroxide solution of SM4 or a potassium hydroxide solution of SM4; in the strong alkaline solution of SM4, the dosage ratio of SM4, strong alkali and water is 1.10g:0.17-0.51g:2-10ml; the pH after acid adjustment is 2-4.
8. The method for preparing a telpotide side chain intermediate according to claim 1, characterized in that: The reactions in steps (1) to (6) were all followed by TCL.
Citation Information
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