Preparation process of pentapeptide side chain of tilpoitide

Abstract

The invention discloses a preparation process of a pentapeptide side chain of tilpoitide, and relates to the technical field of preparation of the pentapeptide side chain of tilpoitide. The preparation process disclosed by the invention comprises the following steps: firstly, preparing a fatty acid pentapeptide side chain crude product from a tirpotide eicosanoic acid side chain TRM1 and Fmoc-Lys-OH under the action of a condensing agent; and mixing the fatty acid pentapeptide side chain crude product, an organic solvent and concentrated hydrochloric acid, heating, dissolving and clarifying, adding activated carbon, carrying out hot filtration, adding crystal seeds into filtrate, cooling, and carrying out suction filtration to obtain a tilpotide pentapeptide side chain refined product. According to the preparation process, the use of other metals for salifying is avoided, and the influence of metal ions is eliminated; and secondly, the crystallization operation is simple and convenient, the obtained product is a white solid, the quality is excellent, the ICH standard is met, and the content of key impurities is lower than 0.10%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tirzepatide preparation, in particular to a preparation process of a tirzepatide pentapeptide side chain. BACKGROUND

[0002] Tirzepatide is a once-weekly subcutaneous injection of GLP-1 / GIP dual receptor agonist developed by Eli Lilly, USA, approved by FDA on May 13, 2022 for the treatment of type 2 diabetes (T2DM). Subsequently, it was approved in the European Union, Japan and other countries. On November 8, 2023, FDA approved it for the treatment of obesity or overweight, on May 15, 2024, it was approved in China for the treatment of type 2 diabetes, and on July 19, 2024, it was approved in China for weight loss indications. As of the third quarter of 2025, tirzepatide global sales were $24.837 billion, and it is expected to become the world's new drug king.

[0003]

[0004] For the preparation of tirzepatide, Eli Lilly mainly discloses two types of preparation processes. One (CN107207576B) is to sequentially couple MBHARink or Sieber resin by solid phase, then cut to obtain tirzepatide crude product, and then purify by high pressure preparation. The other (US20220135639A1) is a combination of solid and liquid phases, i.e. cutting the main chain 39 peptide into several fragments, then synthesizing several fragments by solid phase, and then using liquid phase condensation to obtain tirzepatide crude product, and finally purifying by high pressure preparation.

[0005] Among them, two types of processes involve a key fatty acid pentapeptide side chain, the structure of which is as follows:

[0006]

[0007] Currently, there are mainly the following preparation processes for tirzepatide pentapeptide side chain:

[0008] Shenzhen Shengchuang adopts solid phase synthesis in patent CN112110981 to directly connect one carboxyl group in the double carboxyl monomer at the end of the side chain to the solid phase carrier resin, and then gradually complete the coupling of the entire peptide sequence by using different protected amino acid monomers from the side chain to the two ends of the main chain. However, the three wastes are highly polluting, the crude product has low purity, and the purity is only 70%.

[0009] Sichuan Prukang discloses the purification and preparation method of the side chain in patent CN119431193B, which mainly uses salt crystallization with barium hydroxide, calcium hydroxide and other substances; but introduces new metal ions, which affects the quality control or feed conversion of subsequent solid phase synthesis.

[0010] Jiangsu Nota Australis disclosed a method for preparing a fatty diacid fragment in patent CN 118546077 A. The long-chain fatty diacid with a single tert-butyl group is sequentially condensed with 1-glutamic acid tert-butyl ester, 2-(2-(2-aminoethoxy)ethoxy)acetic acid, 2-(2-(2-aminoethoxy)ethoxy)acetic acid, and Fmoc-lysine to prepare the fatty diacid fragment. The purity of the fatty diacid fragment is above 99.0%. However, active protecting groups such as pentafluorophenol are introduced during the preparation process, increasing the quality research; and the obtained product is concentrated to a dry oil, which is not convenient for feeding and content yield calculation.

[0011] Suzhou Teli Pharmaceutical disclosed a method for preparing a polypeptide drug side chain pentapeptide at low temperature in patent CN 120209075. HOSU or pentafluorophenol is used as a leaving protecting group, and the target product is obtained by ultra-low temperature freezing centrifugation at minus 20 degrees Celsius. Although the product is in solid form, it is obtained by freeze-drying, which requires very high energy consumption and is inconvenient to operate, and is not conducive to industrial production.

[0012] Therefore, finding a suitable industrial production process for preparing a very good solid tipepitide pentapeptide side chain that meets the ICH quality standards is a technical problem that needs to be solved in the field at present.

[0013] In view of this, the present application is proposed. SUMMARY

[0014] The purpose of the present application is to provide a preparation process for a tipepitide fatty acid pentapeptide side chain. The preparation process can greatly reduce the number of preparation column chromatography and the generated wastewater and waste salt, is green and environmentally friendly, has low requirements for equipment, and is simple to operate. Secondly, the experimental steps are simplified, the prepared tipepitide fatty acid pentapeptide side chain finished product is in solid form, which is convenient for storage and pharmaceutical research, and has higher purity, lower single impurity content, and quality that meets the ICH quality requirements (all single impurities are lower than 0.10%).

[0015] In a first aspect, the present application provides a process for preparing a tipepitide pentapeptide side chain, characterized in that it comprises the following steps:

[0016] 1) reacting tipepitide eicosanoic acid side chain TRM1 and Fmoc-Lys-OH under the action of a condensing agent to obtain a tipepitide pentapeptide side chain crude product,

[0017] 2) mixing the tipepitide pentapeptide side chain crude product obtained in step 1), an organic solvent, and concentrated hydrochloric acid, heating to dissolve, adding activated carbon, hot filtering, adding seed crystals to the filtrate, cooling, and suction filtering to obtain a tipepitide pentapeptide side chain finished product,

[0018]

[0019] In some embodiments, the condensing agent is butyl phosphinic anhydride, propyl phosphinic anhydride, or a combination thereof.

[0020] In some embodiments, the molar ratio of TRM1 to condensing agent is 1:(1-5); preferably, the molar ratio of TRM1 to condensing agent is 1:(1.5-4); more preferably, the molar ratio of TRM1 to condensing agent is 1:(2-3.5); further preferably, the molar ratio of TRM1 to condensing agent is 1:(2-3) (e.g., 1:2, 1:2.5, 1:3).

[0021] In some embodiments, the molar ratio of TRM1 to Fmoc-Lys-OH is 1:(1-2); preferably, the molar ratio of TRM1 to Fmoc-Lys-OH is 1:(1-1.8); more preferably, the molar ratio of TRM1 to Fmoc-Lys-OH is 1:(1-1.5); further preferably, the molar ratio of TRM1 to Fmoc-Lys-OH is 1:(1-1.3) (e.g., 1:1.05, 1:1.1, 1:1.15, 1:1.2, 1:1.25, 1:1.3).

[0022] In some embodiments, the organic solvent in step 2) is selected from any one of acetone, methanol, acetonitrile.

[0023] In some embodiments, the mass volume ratio of crude telopeptide pentapeptide side chain to organic solvent in step 2) is 1:(3-10) g / ml; preferably, the mass volume ratio of crude telopeptide pentapeptide side chain to organic solvent is 1:(5-10) g / ml; more preferably, the mass volume ratio of crude telopeptide pentapeptide side chain to organic solvent is 1:(7-8) g / ml; further preferably, the mass volume ratio of crude telopeptide pentapeptide side chain to organic solvent is 1:7.5 g / ml.

[0024] In some embodiments, the mass volume ratio of crude telopeptide pentapeptide side chain to organic solvent is 1:(5-7).

[0025] In some embodiments, the temperature for heating the solution in step 2) is 30-50°C; preferably, the temperature for heating the solution is 40-50°C (e.g., 45°C).

[0026] In some embodiments, the cooling refers to cooling to 0-15°C.

[0027] In some embodiments, the mass ratio of the crude telopeptide fatty acid pentapeptide side chain to concentrated hydrochloric acid is 1:(0.0005-0.005); preferably, the mass ratio of the crude telopeptide fatty acid pentapeptide side chain to concentrated hydrochloric acid is 1:(0.0008-0.003); more preferably, the mass ratio of the crude telopeptide fatty acid pentapeptide side chain to concentrated hydrochloric acid is 1:(0.001-0.002) (e.g. 1:0.001, 1:0.0015, 1:0.002).

[0028] In some embodiments, the amount of activated carbon used is 3%-5% (e.g. 3.5%, 4%, 4.5%, 5%) of the mass of the crude telopeptide fatty acid pentapeptide side chain.

[0029] In some embodiments, the amount of seed crystal used is 0.5%-5% of the mass of the crude pentapeptide side chain; preferably, the amount of seed crystal used is 0.5%-3% of the mass of the crude pentapeptide side chain; more preferably, the amount of seed crystal used is 1%-2% (e.g. 1%, 1.2%, 1.5%, 1.8%, 2%) of the mass of the crude pentapeptide side chain.

[0030] It should be noted that the mass volume ratio described herein refers to g:ml.

[0031] In some embodiments, the HPLC purity of the crude telopeptide fatty acid pentapeptide side chain is 95%-98%; preferably, the HPLC purity of the crude telopeptide fatty acid pentapeptide side chain is 96%-98%; more preferably, the HPLC purity of the crude telopeptide fatty acid pentapeptide side chain is 97%-98% (e.g. 97.1%, 97.14%, 97.15%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%).

[0032] The present application has the following advantages:

[0033] The process provided by the present application first obtains a crude pentapeptide side chain TRM2 by reacting telopeptide eicosanoic acid side chain TRM1 and Fmoc-Lys-OH in the presence of a condensing agent, then mixes the crude pentapeptide side chain, an organic solvent and hydrochloric acid, heats and dissolves, adds activated carbon, hot filters, adds seed crystals to the filtrate, cools, and filters to obtain a refined telopeptide pentapeptide side chain product. This preparation and purification process avoids the use of conventional column chromatography purification, metal ion salt refining or ultra-low temperature freezing crystallization, greatly reduces the waste solvent generated during preparation, has low energy consumption and simple operation; secondly, it simplifies the experimental steps, the prepared product has higher purity, all impurities are less than 0.10%, and the quality is excellent. The telopeptide fatty acid pentapeptide side chain product obtained by the present application is in solid form, which is convenient for storage and pharmaceutical research

[0034] The preparation process provided by this invention avoids the conventional metal ion salt formation, concentration, and low-temperature freezing methods. Instead, it uses a mixed system of organic solvent and hydrochloric acid to dissolve the crude telpoide fatty acid side chain, resulting in a smaller solvent consumption, improved purity, and reduced impurity content. The addition of seed crystals facilitates the precipitation of fatty acid side chain crystals. Activated carbon is used for decolorization or to remove some inorganic impurities. Appropriate cooling can precipitate fatty acid side chain crystals and improve the yield. Attached Figure Description

[0035] Figure 1 The HPLC detection results are as follows: The crude telpopeptide pentapeptide side chain obtained in Example 1 of this invention.

[0036] Figure 2 The HPLC detection results are for the purified product of the telpopeptide pentapeptide side chain obtained in Example 1 of this invention.

[0037] Figure 3 The HPLC detection results are for the crude telpopeptide pentapeptide side chain obtained in Comparative Example 1 of this invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0039] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0040] Example 1

[0041] 1) Preparation of crude telpopeptide pentapeptide side chain

[0042] In a 20L reaction flask, C20-OtBu-Glu(OtBu)-AEEA-AEEA-OH (1500g, 1eq) and Fmoc-Lys-OH (664.2g, 1.05eq) were added sequentially and dissolved in 15L of dichloromethane. The mixture was cooled to 10–15℃, and butyl phosphoric anhydride (1545.5g, 2.5eq) was slowly added dropwise. After the addition was complete, the reaction was allowed to proceed at room temperature for 4–6 hours. The reaction system was then cooled to 0–15℃, and 5L of water was added to quench the reaction. The dichloromethane organic phase was washed sequentially with 0.5N sodium bicarbonate solution (500ml*2) and 0.5N dilute hydrochloric acid (500ml*2), dried over anhydrous sodium sulfate, and concentrated to dryness to obtain the crude pentapeptide side chain product TRM2, approximately 2102g of oily substance (100% yield).

[0043] The HPLC results of crude pentapeptide side chain TRM2 are shown in the figure.Figure 1 The purity is 97.14%.

[0044] Chromatographic conditions

[0045] Chromatographic column: CAPCELL PAKADME-HR, 4.6 mm × 250 mm, 3 μm; Detection wavelength: 200 nm; Flow rate: 1 ml / min; Column temperature: 40 ℃; Injection volume: 5 μl; Injector temperature: 8 ℃.

[0046] Mobile phase A: 0.1% phosphoric acid solution; Mobile phase B: acetonitrile; Test solution: Take an appropriate amount of this product, accurately weigh it, add diluent to dissolve and dilute to prepare a solution containing about 1 mg per 1 ml (e.g., take about 20 mg of this product, accurately weigh it, place it in a 20 ml volumetric flask, add diluent to dissolve and dilute to the mark, and shake well).

[0047] 2) Preparation of the telpopeptide pentapeptide side chain product

[0048] 2000g of the crude telpopeptide pentapeptide side chain obtained above was added to a 20L glass reaction flask, followed by the addition of 15L acetone and 2g of concentrated hydrochloric acid (36% by mass). The mixture was heated to 45℃ to dissolve completely, then 100g of activated carbon was added. The mixture was stirred for 30 minutes and then hot-filtered at this temperature. At 25℃, 20g of seed crystals were added at once, and the mixture was stirred and crystallized for 6–8 hours, resulting in a large amount of solid precipitation. The mixture was then cooled to 0–5℃ and crystallized for 3 hours, followed by filtration. The filter cake was dried under vacuum at room temperature to obtain approximately 1790g of white solid. This is the purified telpopeptide pentapeptide side chain product, with a yield of 89.5%. The HPLC results are shown below. Figure 2 The purity was 99.82%, with all single impurities below 0.10%. MS (ESI) + ): m / z1225.3 [M+H] + .

[0049] Comparative Example 1: Preparation of crude telpopeptide side chain

[0050] TRM1, N,N'-succinimide carbonate (DSC) and dichloromethane (DCM) were added to a reaction flask and stirred at 25-30°C for 16-18 hours. A sample was taken and spotted onto a TLC plate, which showed that a small amount of raw materials remained. The reaction solution was washed twice with 50 ml of water, dried over anhydrous sodium sulfate, and filtered to obtain a dichloromethane solution of TRM1OSu.

[0051] The above-mentioned TRM1OSu DCM solution and Fmoc-Lys-OH were added to the reaction flask and stirred at 25-30℃ for 3-4 hours. TLC detection showed that the reaction was complete.

[0052] Post-processing: The reaction solution was washed three times with 50 ml of 1N HCl and once with 50 ml of 10% NaCl solution. It was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 21 g of oily product, with a yield of 100%; however, the purity was only 95.97%.

[0053] Comparative Example 2: Preparation of the telpopeptide pentapeptide side chain product

[0054] Referring to step (2) in Example 1, without adding concentrated hydrochloric acid, and with the rest of the operation unchanged, no solid was precipitated in the end.

[0055] Comparative Example 3: Preparation of the telpopeptide pentapeptide side chain product

[0056] Referring to step (2) in Example 1, the amount of concentrated hydrochloric acid was increased to 1% (200g substrate, then 2g of concentrated hydrochloric acid was added), and the rest of the operation remained unchanged, but no solid was precipitated in the end.

[0057] In summary, the purification method for high-purity telpopeptide pentapeptide side chains described in the embodiments of the present invention is green and environmentally friendly, and simple to operate; the purified product is a white solid with greatly improved purity and less than 0.10% impurities.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A process for preparing the side chain of telpoeptide pentapeptide, characterized in that, It includes the following steps: 1) The crude pentapeptide side chain of telpopeptide was obtained by reacting the eicosanoic acid side chain TRM1 and Fmoc-Lys-OH with a condensing agent. 2) Mix the crude telpopeptide pentapeptide side chain obtained in step 1), organic solvent, and concentrated hydrochloric acid, heat to dissolve, add activated carbon, hot filter, add seed crystals to the filtrate, cool, and filter under vacuum to obtain the finished telpopeptide pentapeptide side chain.

2. The method for preparing crude telpopeptide side chain according to claim 1, characterized in that, The condensing agent is butyl phosphate anhydride, propyl phosphate anhydride, or a combination thereof.

3. The method for preparing crude telpopeptide pentapeptide side chain according to claim 1 or 2, characterized in that, The organic solvent mentioned in step 2) is selected from any one of acetone, methanol, and acetonitrile.

4. The method for preparing the telpopeptide pentapeptide side chain according to any one of claims 1-3, characterized in that, In step 2), the mass-to-volume ratio of crude telpopeptide pentapeptide side chain to organic solvent is 1:(3-10) g / ml; preferably, the mass-to-volume ratio of crude telpopeptide pentapeptide side chain to organic solvent is 1:(5-10) g / ml; more preferably, the mass-to-volume ratio of crude telpopeptide pentapeptide side chain to organic solvent is 1:(7-8) g / ml; and even more preferably, the mass-to-volume ratio of crude telpopeptide pentapeptide side chain to organic solvent is 1:7.5 g / ml.

5. The method for preparing the telpopeptide pentapeptide side chain according to any one of claims 1-4, characterized in that, The heating temperature for dissolving the liquid in step 2) is 30–50°C; preferably, the heating temperature for dissolving the liquid is 40–50°C.

6. The method for preparing the telpopeptide pentapeptide side chain according to any one of claims 1-5, characterized in that, The mass ratio of crude telpopeptide side chain to concentrated hydrochloric acid is 1:(0.0005-0.005); preferably, the mass ratio of crude telpopeptide side chain to concentrated hydrochloric acid is 1:(0.0008-0.003); more preferably, the mass ratio of crude telpopeptide side chain to concentrated hydrochloric acid is 1:(0.001-0.002).

7. The method for preparing the telpopeptide pentapeptide side chain according to any one of claims 1-6, characterized in that, The amount of activated carbon used is 3% to 5% of the crude mass of the telpopeptide pentapeptide side chain.

8. The method for preparing the telpopeptide pentapeptide side chain according to any one of claims 1-8, characterized in that, The amount of seed crystals used is 0.5% to 5% of the crude pentapeptide side chain mass; preferably, the amount of seed crystals used is 0.5% to 3% of the crude pentapeptide side chain mass; more preferably, the amount of seed crystals used is 1% to 2% of the crude pentapeptide side chain mass.

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