Purification Method and Preparation Method of Long-Acting Side Chain

Abstract

The present invention relates to the technical field of drug purification, and in particular, to a purification method of a long-acting side chain and a preparation method thereof. In the embodiments of the present invention, by controlling the pH and extraction and then solidifying and crystallizing by salting, a crystalline substance in the form of a salt with a higher purity is obtained. After desalting, extraction, and washing to remove salts, a free long-acting side chain is obtained, making the long-acting side chain have a higher purity and a low impurity content. At the same time, by using the purification method in the embodiments of the present invention, the generation amount of three wastes such as waste organic solvents, waste water, and solid waste can be reduced, and the production cost is saved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug purification, and more particularly, to a purification method for a long-acting side chain and a preparation method thereof. Background Art

[0002] Diabetes is a metabolic disorder characterized by long-term hyperglycemia and is one of the most common chronic diseases globally. Human glucagon-like peptide-1 (GLP-1) is an effective drug for treating diabetes.

[0003] The side chains of semaglutide and tirzepatide are long-acting side chains of human glucagon-like peptide-1 (GLP-1) analogs. These side chain compounds are protected amino acids with Fmoc, which can be coupled with polypeptides to form semaglutide and tirzepatide.

[0004] Since the above long-acting side chains are liquids or pastes at room temperature, the current purification process is mainly column chromatography, which generates a large amount of three wastes. Moreover, the special requirements of the chromatography equipment not only limit the single-batch production volume but also increase the normal production cycle, posing a huge challenge to the large-scale production of products.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a purification method for a long-acting side chain and a preparation method thereof. The purification method provided by the embodiments of the present invention can effectively remove impurities in the crude long-acting side chain, improve the purity of the long-acting side chain. At the same time, it can reduce the generation of three wastes such as waste mailing solvents and solid wastes, save production costs, and shorten the production cycle.

[0007] The present invention is implemented as follows:

[0008] In a first aspect, the present invention provides a purification method for a long-acting side chain, including: mixing the crude long-acting side chain with a mixed solvent and then adjusting the pH to 3.1 - 3.5; wherein, the mixed solvent is a mixture of water and epoxy ether;

[0009] Then, extracting with C7 - C10 alkanes and chloromethane in sequence and collecting the first aqueous phase,

[0010] Next, mixing the first aqueous phase with sodium chloride, adjusting the pH to 2.5 - 2.8, and then extracting with the chloromethane to collect the second aqueous phase and the first oil phase,

[0011] Then, adjusting the pH of the second aqueous phase to 2.1 - 2.4, and extracting with the chloromethane to collect the second oil phase;

[0012] Next, combining the first oil phase and the second oil phase, and drying and concentrating to form a refined long-acting side chain product 1;

[0013] Then, the refined long-acting side chain 1 is mixed and stirred with a salifying substance to form a salt, and then a crystallization solvent and seeds are added for crystallization to form the refined long-acting side chain 2;

[0014] Then, the refined long-acting side chain 2 is mixed with chloromethane and acid brine for desalting, and after phase separation, the third aqueous phase is collected. The third aqueous phase is extracted with chloromethane and the third oil phase is collected.

[0015] In an alternative embodiment, the epoxy ether is selected from tetrahydrofuran, 2-methyltetrahydrofuran, and 1,4-dioxane;

[0016] Preferably, 3 - 4 kg of water and 0.2 - 0.3 kg of the epoxy ether are added per kg of the crude long-acting side chain.

[0017] In an alternative embodiment, the C7-C10 alkane is selected from n-heptane and n-decane;

[0018] Preferably, the chloromethane is selected from dichloromethane or trichloromethane.

[0019] In an alternative embodiment, the number of extractions with the C7-C10 alkane and the chloromethane is 1 - 3 times respectively.

[0020] In an alternative embodiment, 0.3 - 0.4 kg of sodium chloride is added per kg of the crude long-acting side chain.

[0021] In an alternative embodiment, the salifying substance is selected from barium hydroxide and calcium hydroxide;

[0022] Preferably, the crystallization solvent is selected from C7-C10 alkanes, more preferably n-heptane and n-decane.

[0023] In an alternative embodiment, the step of forming the refined long-acting side chain 2 includes: mixing the refined long-acting side chain 1 with the salifying substance and stirring at 10 - 30 °C for 1 - 2 h, then adding the C7-C10 alkane and seeds and continuing to stir until a solid precipitates, and then adding the C7-C10 alkane and continuing to stir for 2 - 3 h.

[0024] In an alternative embodiment, the acid brine includes concentrated hydrochloric acid, water, and sodium chloride;

[0025] Preferably, the concentration of the acid brine is 0.8 - 1.2%.

[0026] In an alternative embodiment, the crude long-acting side chain includes the crude side chain of semaglutide and the crude side chain of tirzepatide.

[0027] Second aspect, the present invention provides a method for preparing a long-acting side chain, which includes the purification method of the long-acting side chain described in any one of the foregoing embodiments.

[0028] The present invention has the following beneficial effects: In the embodiments of the present invention, by controlling the pH and extraction and then solidifying and crystallizing by salting, a crystalline substance in the form of a salt with higher purity is obtained. After desalting, extraction, and washing to remove salts, a free long-acting side chain is obtained, making the long-acting side chain have higher purity and low impurity content. At the same time, by using the purification method in the embodiments of the present invention, the generation amount of the three wastes such as waste organic solvents, waste water, and solid waste can be reduced, the production cost is saved, and the production cycle is shortened. For example, the existing extraction washing + column chromatography can be used. Detailed Embodiments

[0029] 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. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0030] First aspect, the present invention provides a purification method for a long-acting side chain. The long-acting side chain includes the semaglutide side chain and the tirzepatide side chain, and the specific structural formula is as follows:

[0031] The impurities contained therein include the bis-Lys(Fmoc) impurity, the 3AEEA impurity, the mono-AEEA impurity, the de-Fmoc impurity, and the de-tert-butyl impurity, and the specific structural formulas are as follows:

[0032]

[0033] The inventors found through research that there is an acidic difference between the above impurities and the long-acting side chain. Then, the long-acting side chain can be separated from the above impurities by adjusting the pH and extraction, improving the purity of the long-acting side chain, and reducing the content of each impurity.

[0034] Specifically, the crude long-acting side chain is mixed and stirred with a mixed solvent. Among them, the crude long-acting side chain is prepared with reference to CN114805543A. The mixed solvent is selected from the mixture of water and epoxy ether; 3-4 kg of water and 0.2-0.3 kg of the epoxy ether are added per kg of the crude long-acting side chain. The epoxy ether is selected from tetrahydrofuran, 2-methyltetrahydrofuran, and 1,4-dioxane.

[0035] Using the above mixed solvent can dissolve the long-acting side chain and impurities, which is beneficial to subsequent separation.

[0036] Then, the pH is adjusted to 3.1 - 3.5 with an acid, such as 3.1, 3.2, 3.3, 3.4, 3.5, or any value between 3.1 and 3.5. The acid used for pH adjustment is an acidic substance, such as hydrochloric acid.

[0037] Then, the above solution is extracted with a C7 - C10 alkane to collect the aqueous phase, so as to remove epoxy ethers and small - polarity impurities. Among them, the C7 - C10 alkane is selected from n - heptane and n - decane. For each extraction, 0.5 - 0.7 kg of the C7 - C10 alkane is added per kg of the crude product of the long - acting side chain.

[0038] The above aqueous phase is further extracted with chloromethane and the first aqueous phase is collected. The chloromethane is selected from dichloromethane or trichloromethane. For each extraction, 3 - 4 kg of chloromethane is added per kg of the crude product of the long - acting side chain.

[0039] The first aqueous phase is mixed with sodium chloride and then the pH is adjusted to 2.5 - 2.8. Specifically, the first aqueous phase and sodium chloride are stirred, and then the pH is adjusted to 2.5 - 2.8 with an acid. 0.3 - 0.4 kg of sodium chloride is added per kg of the crude product of the long - acting side chain.

[0040] The addition of sodium chloride can accelerate the separation of organic substances, improve the separation efficiency, and reduce the loss of products.

[0041] Then, chloromethane extraction is added to the above solution for extraction, and the second aqueous phase and the first oil phase are collected respectively. The chloromethane is dichloromethane or trichloromethane, and 3 - 4 kg of chloromethane is added per kg of the crude product of the long - acting side chain for each extraction.

[0042] Next, the pH of the second aqueous phase is adjusted to 2.1 - 2.4, chloromethane extraction is added to the above solution for extraction, and the second oil phase is collected. The chloromethane is dichloromethane or trichloromethane, and 3 - 4 kg of chloromethane is added per kg of the crude product of the long - acting side chain for each extraction.

[0043] Then, the first oil phase and the second oil phase are combined, that is, the chloromethane phases extracted at pH = 2.5 - 2.8 and 2.1 - 2.4 are combined, and dried and concentrated to form the refined product 1 of the long - acting side chain; specifically, anhydrous magnesium sulfate is added for drying, filtered and concentrated to obtain the refined product 1 of the long - acting side chain.

[0044] Mix the refined long-acting side chain 1 with a salifying substance and stir to form a salt, then add a crystallization solvent and crystal seeds for crystallization to form refined long-acting side chain 2. Specifically, mix the refined long-acting side chain 1 with an epoxy ether, dropwise add the salifying substance, stir at 10 - 30 °C for 1 - 2 h, then add the C7 - C10 alkane and crystal seeds and continue stirring until a solid precipitates, and then add the C7 - C10 alkane and continue stirring for 2 - 3 h. A large amount of solid (salt) precipitated is the refined long-acting side chain 2. The above salifying substance includes barium hydroxide and calcium hydroxide; the C7 - C10 alkane is n - heptane and n - decane.

[0045] Then filter and wash to obtain a solid. Mix the solid with chlorinated methane and acid brine for desalting and perform phase separation, then collect the third aqueous phase. Specifically, add chlorinated methane (3.5 - 4.5 times the mass of the crude long-acting side chain) to the solid, then add acid brine and stir, perform phase separation, collect the third aqueous phase, and then add chlorinated methane to extract the third aqueous phase and collect the third oil phase. The chlorinated methane includes dichloromethane or trichloromethane. Each time during extraction, 0.5 - 0.7 kg of chlorinated methane is added per kg of the crude long-acting side chain.

[0046] The acid brine includes concentrated hydrochloric acid, water, and sodium chloride; the concentration of the acid brine is 0.8 - 1.2%.

[0047] The number of extractions described in the embodiments of the present invention is 1 - 3 times.

[0048] Then, dry and concentrate the third oil phase. For example, dry with anhydrous magnesium sulfate and filter and concentrate to obtain the long-acting side chain.

[0049] In a second aspect, the present invention provides a preparation method of a long-acting side chain, which includes the purification method of the long-acting side chain according to any one of the foregoing embodiments.

[0050] It should be noted that the purity detection method provided in the embodiments of the present invention is as follows:

[0051] Chromatographic column: Waters X - Bridge C18 (150 * 4.6 mm, 3.5 μm) or a chromatographic column with equivalent performance;

[0052] Trapping column: NanoChrom Ghost - Remover, 4.6 × 50 mm;

[0053] Mobile phase: 0.1% perchloric acid aqueous solution as mobile phase A, 95% acetonitrile aqueous solution as mobile phase B;

[0054] Injection volume: 10 μl;

[0055] Flow rate: 0.8 ml / min;

[0056] Detection wavelength: 210 nm.

[0057] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0058] Example 1

[0059] The embodiment of the present invention provides a purification method for a long-acting side chain, including:

[0060] 15 kg of the crude semaglutide side chain (prepared with reference to CN114805543A, with a purity of 91.86% for this crude product) is added to 50 kg of water and 4 kg of tetrahydrofuran, stirred evenly, and the pH is adjusted to 3.1 with 4 mol / L hydrochloric acid.

[0061] N-heptane is added for extraction twice (2 * 10 kg) to remove tetrahydrofuran and small-polarity impurities.

[0062] The aqueous phase is further extracted with dichloromethane for impurities twice (2 * 45 kg).

[0063] After extracting impurities, 5 kg of sodium chloride is first added to the aqueous phase; stirred, the pH is adjusted to 2.5 with 4 mol / L hydrochloric acid, and dichloromethane is added for extraction twice (2 * 45 kg).

[0064] The aqueous phase after extraction is further adjusted to pH = 2.1 with 4 mol / L hydrochloric acid, and dichloromethane is added for extraction twice (2 * 45 kg).

[0065] The dichloromethane phases extracted four times at pH = 2.5 and 2.1 are combined, dried with anhydrous magnesium sulfate, filtered and concentrated to obtain the refined long-acting side chain 1.

[0066] To the refined product 1, 20 kg of tetrahydrofuran is added, an aqueous solution of Ba(OH)₂ is added dropwise, and stirred at 30 °C for 1 h; 20 kg of n-heptane is added dropwise; after adding seed crystals and stirring until solids precipitate, 20 kg of n-heptane is continuously added dropwise, and stirred for 3 h to precipitate a large amount of solids (salts), which is the refined long-acting side chain 2.

[0067] Filtered and washed with n-heptane, 60 kg of dichloromethane is added to the obtained solid, 10 kg of 1% brine (3 kg of concentrated hydrochloric acid + 297 kg of water + 15 kg of sodium chloride) is slowly added, stirred for 2 h after addition, separated, the aqueous phase is further extracted with dichloromethane once (1 * 10 kg), the dichloromethane phases extracted twice are combined, dried with anhydrous magnesium sulfate, filtered and concentrated to obtain the semaglutide side chain finished product.

[0068] The purity of the semaglutide side chain finished product is 99.66%, the contents of its single AEEA and 3AEEA impurities are 0.034% and 0.015% respectively, and the methyl ester derivative and the bis-Fmoc-lys derivative are not detected.

[0069] Example 2

[0070] 15 kg of crude telotristat side chain (prepared with reference to CN114805543A, with a purity of 89.32% for this crude product) was added to 55 kg of water and 4.2 kg of tetrahydrofuran, stirred evenly, and the pH was adjusted to 3.2 with 4 mol / L hydrochloric acid.

[0071] It was extracted twice with n - heptane (2 * 10 kg) to remove tetrahydrofuran and small - polarity impurities.

[0072] The aqueous phase was further extracted twice with dichloromethane (2 * 45 kg) to remove impurities.

[0073] After the aqueous phase was extracted to remove impurities, 5 kg of sodium chloride was first added; stirred, the pH was adjusted to 2.8 with 4 mol / L hydrochloric acid, and then extracted twice with dichloromethane (2 * 45 kg).

[0074] The aqueous phase after extraction was further adjusted to pH = 2.4 with 4 mol / L hydrochloric acid and extracted twice with dichloromethane (2 * 45 kg).

[0075] The dichloromethane phases extracted four times at pH = 2.8 and 2.4 were combined, dried with anhydrous magnesium sulfate, filtered and concentrated to obtain refined long - acting side chain 1.

[0076] To the refined product 1, 22 kg of tetrahydrofuran was added, an aqueous solution of Ba(OH)₂ was added dropwise, and stirred at 26 °C for 1.5 h; 20 kg of n - heptane was added dropwise; after adding seed crystals and stirring until solids precipitated, 20 kg of n - heptane was continuously added dropwise and stirred for 3 h, and a large amount of solids (salts) precipitated, which was refined product 2.

[0077] After filtration and washing with n - heptane, 60 kg of dichloromethane was added to the obtained solid, 10 kg of 1.1% brine (3.3 kg of concentrated hydrochloric acid + 297 kg of water + 15 kg of sodium chloride) was slowly added, and after adding, it was stirred for 2 h, phase - separated, the aqueous phase was further extracted once with dichloromethane (1 * 10 kg), the dichloromethane phases extracted twice were combined, dried with anhydrous magnesium sulfate, filtered and concentrated to obtain the finished product of telotristat side chain.

[0078] The purity of the finished product of telotristat side chain was 99.66%, the contents of its impurities AEEA and methyl ester derivatives were 0.006% and 0.033% respectively, and bis - Fmoc - lys and 3AEEA were not detected.

[0079] Process Optimization 1

[0080] Referring to the purification method provided in Example 1, refined long - acting side chain 2 was prepared using refined long - acting side chain 1. The steps of this method were the same as those in Example 1, except that the salting - out substance was different. See Table 1 below for details.

[0081] Table 1 Salt-forming substances

[0082]

[0083]

[0084] It can be seen that if other salt-forming substances are changed, it is simply impossible to precipitate solids, that is, it is impossible to form the long-acting side-chain refined product 2. Although calcium hydroxide can precipitate solids, since it is a slurry solid, a large amount of it remains in the production kettle during large-scale production, resulting in the accumulation of materials in the kettle and the need to regularly clean the production kettle, which reduces the production efficiency. However, using barium hydroxide provided in the embodiment of the present invention can not only obtain the long-acting side-chain refined product 2 with high purity but also ensure continuous production and improve production efficiency.

[0085] Process optimization 2

[0086] Prepare the long-acting side-chain refined product 1 by referring to the purification method provided in Example 1. The specific method and process are all referred to Example 1, and the only difference is the pH value. See Table 2 for details.

[0087] Table 2 Selection of pH

[0088]

[0089] As can be seen from Table 2 above, by changing the pH value, different impurities are extracted and removed at different pH values. The product can be basically extracted when the pH is between 2.0 and 3.0, and the remaining extracted impurities can be removed by subsequent salt formation to achieve the purpose of purifying the finished product and reducing losses.

[0090] Process optimization 3

[0091] This process provides a method for an existing purification process, which is as follows:

[0092] Take 15 kg of the crude semaglutide side chain and add 18 kg of ethyl acetate and stir to dissolve.

[0093] Load 60 kg of silica gel into a column, add 75 kg of n-heptane to soak the column until it is completely wetted by n-heptane; load the sample.

[0094] First, use ethyl acetate - n-heptane (mass ratio = 2:1) as the eluent to wash away impurities.

[0095] Then use dichloromethane - methanol (mass ratio = 25:1) as the eluent to elute the product.

[0096] The product phase is concentrated at 40 - 45 °C to obtain the finished semaglutide side chain.

[0097] Among them, the crude product of the side chain of semaglutide was prepared with reference to CN114805543A. The organic waste liquid generated by this process was 22,600 kg, the solid waste was 675 kg, and the wastewater was 3,000 kg.

[0098] The crude product of the side chain of semaglutide, which is the same as that in Process Optimization 3, was treated with reference to the purification method provided in Example 1 of the present invention. However, its organic waste liquid was 4,290 kg, the solid waste was 90 kg, and the wastewater was 1,050 kg.

[0099] Process Optimization 4

[0100] With reference to the purification method provided in Example 1, the long-acting side chain crude product was used to prepare the long-acting side chain refined product 1. The steps of this method were the same as those in Example 1, except that the solvents used for extracting impurities after adjusting the pH to 3.1 were different. See Table 3 below for details.

[0101] Table 3 Solvent Selection

[0102] Solvent Selection Extracted Substances n-Decane Large Amounts of Impurities n-Heptane Large Amounts of Impurities n-Hexane No Product, but Less Impurities Extracted than n-Heptane n-Pentane No Product, but Less Impurities Extracted than n-Heptane Methyl tert-Butyl Ether Large Amounts of Impurities, with a Small Amount of Product Extracted Ethyl Acetate Large Amounts of Impurities, with Product Extracted Dichloromethane Large Amounts of Impurities, with More Product Extracted

[0103] When using different solvents to extract small-polarity impurities, n-heptane and n-decane have the best impurity removal effect.

[0104] Process Optimization 5

[0105] With reference to the purification method provided in Example 1, the long-acting side chain crude product was used to prepare the long-acting side chain refined product 1. The steps of this method were the same as those in Example 1, except that the solvents used for extracting the product after adjusting the pH to 2.1 and 2.5 were different. See Table 4 below for details.

[0106] Table 4 Solvent Selection

[0107] Solvent Selection Extracted Substances Dichloromethane Large Amounts of Product, Almost No Impurities Chloroform Large Amounts of Product, Almost No Impurities Ethyl Acetate Large Amounts of Product, Very Little Impurities Methyl Acetate Large Amounts of Product, Very Little Impurities Methyl tert-Butyl Ether Less Amount of Product, No Impurities

[0108] When dichloromethane is used to extract the product, it can reduce the product loss and the amount of solvent used; and at the same time of extracting the product, it can also separate the impurities and the product as much as possible, ensuring the quality of the finished product.

[0109] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A purification method for a long-acting side chain, characterized in that, It includes: Mix the crude long-acting side chain with a mixed solvent and adjust the pH to 3.1 - 3.5; wherein, the mixed solvent is a mixture of water and an epoxy ether; the epoxy ether is selected from tetrahydrofuran, 2-methyltetrahydrofuran, and 1,4-dioxane; Then, extract successively with a C7 - C10 alkane and chloromethane and collect the first aqueous phase, and the chloromethane is selected from dichloromethane or trichloromethane; Next, mix the first aqueous phase with sodium chloride and adjust the pH to 2.5 - 2.8, then extract with the chloromethane and collect the second aqueous phase and the first oil phase, Then, adjust the pH of the second aqueous phase to 2.1 - 2.4, extract with the chloromethane and collect the second oil phase; Next, combine the first oil phase and the second oil phase, and dry and concentrate to form the refined long-acting side chain 1; Then, mix the refined long-acting side chain 1 with a salifying substance and stir to form a salt, then add a crystallization solvent and crystal seeds for crystallization to form the refined long-acting side chain 2; the salifying substance is selected from barium hydroxide and calcium hydroxide; the crystallization solvent is selected from C7 - C10 alkanes; Then, mix the refined long-acting side chain 2 with chloromethane and acid brine for desalting and phase separation, then collect the third aqueous phase, and extract the third aqueous phase with chloromethane and collect the third oil phase; Among them, the long-acting side chain is selected from the semaglutide side chain and the tirzepatide side chain.

2. The purification method of the long-acting side chain according to claim 1, wherein, For each kilogram of the crude long-acting side chain, 3 - 4 kilograms of water and 0.2 - 0.3 kilograms of the epoxy ether are added correspondingly.

3. The purification method of the long-acting side chain according to claim 1, characterized in that, The C7 - C10 alkane is selected from n-heptane and n-decane.

4. The purification method of the long-acting side chain according to claim 1, wherein, The number of extraction times for the C7 - C10 alkane and the chloromethane is 1 - 3 times respectively.

5. The purification method of the long-acting side chain according to claim 1, wherein For each kilogram of the crude long-acting side chain, 0.3 - 0.4 kilograms of sodium chloride are added correspondingly.

6. The purification method of the long-acting side chain according to claim 1, characterized in that, The crystallization solvent is n-heptane and n-decane.

7. The purification method of the long-acting side chain according to claim 6, wherein The step of forming the refined long-acting side chain 2 includes: mixing the refined long-acting side chain 1 with the salifying substance and stirring at 10 - 30 °C for 1 - 2 h, then adding the C7 - C10 alkane and crystal seeds and stirring until a solid precipitates, and then adding the C7 - C10 alkane and continuing to stir for 2 - 3 h.

8. The purification method of the long-acting side chain according to claim 1, wherein The acid brine includes concentrated hydrochloric acid, water, and sodium chloride.

9. The purification method of the long-acting side chain according to claim 8, wherein The concentration of the acid brine is 0.8 - 1.2%.

10. A preparation method of a long-acting side chain, characterized in that, It includes the purification method of the long-acting side chain according to any one of claims 1 - 9.

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