Purification method and preparation method of long-acting side chain

Abstract

The invention relates to the technical field of medicine refining, in particular to a purification method and a preparation method of a long-acting side chain. According to the embodiment of the invention, the long-acting side chain is solidified and crystallized by controlling the pH value, extracting and salifying to obtain the crystal substance in a salt form with higher purity, and then the long-acting side chain in a free state is obtained after salt dissolving, extracting, washing and desalting, so that the long-acting side chain is higher in purity and low in impurity content. Meanwhile, by adopting the purification method provided by the embodiment of the invention, the output of three wastes such as waste organic solvents, wastewater and solid wastes 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 in particular to a purification method and a preparation method of a long-acting side chain. Background Art

[0002] Diabetes is a metabolic disorder characterized by long-term high blood sugar levels and is one of the most common chronic diseases in the world. Human glucagon-1 (GLP-1) is an effective drug for the treatment of diabetes.

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

[0004] However, since the above-mentioned long-acting side chains are liquid or paste-like at room temperature, the current purification process is basically based on column chromatography, which produces a huge amount of three wastes. In addition, the special requirements of chromatography equipment not only limit its single-batch production volume, but also increase the normal production cycle, bringing huge challenges to the large-scale production of products.

[0005] In view of this, the present invention is 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 in the embodiment of the present invention can effectively remove impurities in the crude long-acting side chain and improve the purity of the long-acting side chain. At the same time, it can reduce the generation of waste solvents, solid waste and other three wastes, save production costs, and shorten the production cycle.

[0007] The present invention is achieved in that:

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

[0009] Then, C7-C10 alkane and methyl chloride are used to extract and the first aqueous phase is collected.

[0010] Next, the first aqueous phase is mixed with sodium chloride and the pH is adjusted to 2.5-2.8, and then the second aqueous phase and the first oil phase are extracted with the methyl chloride and collected.

[0011] Then, the pH of the second aqueous phase is adjusted to 2.1-2.4, and the second oil phase is extracted with the methyl chloride and collected;

[0012] Next, the first oil phase and the second oil phase are combined, dried and concentrated to form a long-acting side chain refined product 1;

[0013] Then, the long-acting side chain refined product 1 is mixed with a salt-forming substance and stirred to form a salt, and then a crystallization solvent and a seed crystal are added to perform crystallization to form a long-acting side chain refined product 2;

[0014] Then, the long-acting side chain refined product 2 is mixed with methyl chloride and salt water for desalting and phase separation, and then the third aqueous phase is collected. The third aqueous phase is extracted with methyl chloride and the third oil phase is collected.

[0015] In an optional 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 optional embodiment, the C7-C10 alkane is selected from n-heptane and n-decane;

[0018] Preferably, the methyl chloride is selected from dichloromethane or chloroform.

[0019] In an optional embodiment, the C7-C10 alkane and the methyl chloride are extracted 1-3 times respectively.

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

[0021] In an optional embodiment, the salt-forming 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 optional embodiment, the step of forming the long-acting side chain refined product 2 includes: stirring the long-acting side chain refined product 1 and the salt-forming substance at 10-30°C for 1-2h, then adding the C7-C10 alkane and seed crystals and stirring until solid precipitates, and then adding C7-C10 alkane and continuing to stir for 2-3h.

[0024] In an optional embodiment, the acid salt water comprises concentrated hydrochloric acid, water and sodium chloride;

[0025] Preferably, the concentration of the salt water is 0.8-1.2%.

[0026] In an optional embodiment, the long-acting side chain crude product includes semaglutide side chain crude product and telpotide side chain crude product.

[0027] In a second aspect, the present invention provides a method for preparing a long-acting side chain, which includes the method for purifying the long-acting side chain described in any one of the aforementioned embodiments.

[0028] The present invention has the following beneficial effects: the embodiment of the present invention controls pH and extracts and then solidifies and crystallizes by salt formation to obtain a crystal in the form of a salt with a higher purity, and then obtains a free long-acting side chain after desalting, extraction, washing and desalting, so that the long-acting side chain has a higher purity and a low impurity content. At the same time, the purification method of the embodiment of the present invention can reduce the generation of three wastes such as waste organic solvents, waste water and solid waste, save production costs, and shorten the production cycle. For example, the existing extraction washing + column chromatography can be used. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0030] In the first aspect, the present invention provides a method for purifying a long-acting side chain, wherein the long-acting side chain includes a semaglutide side chain and a telpotide side chain, and the specific structural formula is as follows:

[0031] The impurities contained therein include di-Lys(Fmoc) impurities, 3AEEA impurities, single AEEA impurities, de-Fmoc impurities and de-tert-butyl impurities. The specific structural formula is as follows:

[0032]

[0033] The inventors have found through research that there is a difference in acidity between the above impurities and the long-acting side chains. Therefore, the long-acting side chains can be separated from the above impurities through pH adjustment and extraction, thereby improving the purity of the long-acting side chains and reducing the content of each impurity.

[0034] Specifically, the crude long-acting side chain product is mixed with a mixed solvent, wherein the crude long-acting side chain product is prepared with reference to CN114805543A. The mixed solvent is selected from a 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 product. The epoxy ether is selected from tetrahydrofuran, 2-methyltetrahydrofuran and 1,4-dioxane.

[0035] The use of the above-mentioned mixed solvent can dissolve the long-lasting side chains and impurities, which is beneficial for subsequent separation.

[0036] Then, the pH is adjusted to 3.1-3.5 by using an acid, for example, any value between 3.1, 3.2, 3.3, 3.4 and 3.5, etc. The pH is adjusted by using an acidic substance, for example, hydrochloric acid.

[0037] The solution is then extracted with C7-C10 alkane to collect the aqueous phase to remove epoxy ether and small polar impurities. The C7-C10 alkane is selected from n-heptane and n-decane. During each extraction, 0.5-0.7 kg of C7-C10 alkane is added for each kg of the crude long-acting side chain.

[0038] The aqueous phase is further extracted with methyl chloride and the first aqueous phase is collected, wherein the methyl chloride is selected from dichloromethane or chloroform. During each extraction, 3-4 kg of methyl chloride is added for each kg of the crude long-acting side chain product.

[0039] The first aqueous phase is mixed with sodium chloride and 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 acid. 0.3-0.4 kg of sodium chloride is added for each kg of crude long-acting side chain.

[0040] The use of sodium chloride can accelerate the separation of organic matter, improve separation efficiency and reduce product loss.

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

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

[0043] Then, the first oil phase and the second oil phase are combined, that is, the methyl chloride phases obtained by extraction at pH = 2.5-2.8 and 2.1-2.4 are combined, and dried and concentrated to form a long-acting side chain refined product 1; specifically, anhydrous magnesium sulfate is added for drying, filtering and concentration to obtain a long-acting side chain refined product 1.

[0044] The long-acting side chain refined product 1 is mixed with a salt-forming substance and stirred to form a salt, and then a crystallization solvent and a seed are added to crystallize to form a long-acting side chain refined product 2. Specifically, the long-acting side chain refined product 1 is mixed with an epoxy ether, and a salt-forming substance is added dropwise, and stirred at 10-30°C for 1-2h, and then the C7-C10 alkane and the seed are added and stirred again until a solid precipitates, and then the C7-C10 alkane is added and stirred for 2-3h, and a large amount of solid (salt) precipitates, which is the long-acting side chain refined product 2. The above-mentioned salt-forming substance includes barium hydroxide and calcium hydroxide; the C7-C10 alkane is n-heptane and n-decane.

[0045] Then filter and rinse to obtain a solid. Mix the solid with methyl chloride and acid salt water to desalt and separate the phases to collect the third aqueous phase. Specifically, add methyl chloride (3.5-4.5 times the quality of the crude long-acting side chain) to the solid, then add acid salt water to stir, separate the phases, collect the third aqueous phase, then add methyl chloride to extract the third aqueous phase and collect the third oil phase. The methyl chloride includes dichloromethane or chloroform. During each extraction, 0.5-0.7 kg of methyl chloride is added for every kg of the crude long-acting side chain.

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

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

[0048] Then, the third oil phase is dried and concentrated, for example, by drying with anhydrous magnesium sulfate and filtering and concentrating to obtain the long-acting side chain.

[0049] In a second aspect, the present invention provides a method for preparing a long-acting side chain, which includes the method for purifying the long-acting side chain described in any one of the aforementioned embodiments.

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

[0051] Chromatographic column: Waters X-Bridge C18 (150*4.6mm, 3.5μm) or a 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: 0.8ml / min;

[0056] Detection wavelength: 210nm.

[0057] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0058] Example 1

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

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

[0061] Add n-heptane and extract twice (2*10kg) to remove tetrahydrofuran and small polar impurities.

[0062] Dichloromethane was added to the aqueous phase to extract impurities twice (2*45kg).

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

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

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

[0066] To refined product 1, add 20 kg of tetrahydrofuran, drop an aqueous solution of Ba(OH)2, and stir at 30°C for 1 h; drop 20 kg of n-heptane; add seed crystals and stir until solid precipitates, then continue to drop 20 kg of n-heptane and stir for 3 h. A large amount of solid (salt) precipitates, which is the long-acting side chain refined product 2.

[0067] Filter and rinse with n-heptane, add 60 kg of dichloromethane to the obtained solid, slowly add 10 kg of 1% acid salt water (3 kg of concentrated hydrochloric acid + 297 kg of water + 15 kg of sodium chloride), stir for 2 hours after addition, separate the phases, extract the aqueous phase with dichloromethane once (1*10 kg), combine the two extracted dichloromethane phases, add anhydrous magnesium sulfate to dry, filter and concentrate to obtain the finished product of semaglutide side chain.

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

[0069] Example 2

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

[0071] Add n-heptane and extract twice (2*10 kg) to remove tetrahydrofuran and small polar impurities.

[0072] Dichloromethane was added to the aqueous phase to extract impurities twice (2*45kg).

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

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

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

[0076] To refined product 1, add 22 kg of tetrahydrofuran, drop an aqueous solution of Ba(OH)2, and stir at 26°C for 1.5 h; drop 20 kg of n-heptane; add seed crystals and stir until solid precipitates, then continue to drop 20 kg of n-heptane and stir for 3 h. A large amount of solid (salt) precipitates, which is refined product 2.

[0077] Filter and rinse with n-heptane, add 60 kg of dichloromethane to the obtained solid, slowly add 10 kg of 1.1% acid salt water (3.3 kg of concentrated hydrochloric acid + 297 kg of water + 15 kg of sodium chloride), stir for 2 hours after the addition, separate the phases, extract the aqueous phase with dichloromethane once (1*10 kg), combine the two extracted dichloromethane phases, add anhydrous magnesium sulfate to dry, filter and concentrate to obtain the finished product of the tepoxetine side chain.

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

[0079] Process Optimization 1

[0080] Referring to the purification method provided in Example 1, the long-acting side chain refined product 2 was prepared using the long-acting side chain refined product 1. The steps of the method are the same as those of Example 1, and the only difference is that the salt-forming substance is 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, solids cannot be precipitated at all, that is, long-acting side chain refined product 2 cannot be formed. Although solids can be precipitated by using calcium hydroxide, since it is a slurry solid, a large amount of it will remain in the production kettle during scale-up production, resulting in accumulation of materials in the kettle, requiring regular cleaning of the production kettle, and reducing production efficiency. However, the use of barium hydroxide provided in the embodiment of the present invention can not only obtain high-purity long-acting side chain refined product 2 but also ensure continuous production and improve production efficiency.

[0085] Process Optimization 2

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

[0087] Table 2 pH selection

[0088]

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

[0090] Process Optimization 3

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

[0092] Take 15 kg of crude semaglutide side chain and add 18 kg of ethyl acetate to dissolve by stirring;

[0093] 60 kg of silica gel is loaded into the column, and 75 kg of n-heptane is added to soak the column until it is completely wetted with n-heptane; load the sample.

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

[0095] The product was then eluted using dichloromethane-methanol (mass ratio = 25:1) as the eluent.

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

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

[0098] The same crude semaglutide side chain product as process optimization 3 was treated by the purification method provided in Example 1 of the present invention, but the organic waste liquid was 4290 kg, the solid waste was 90 kg, and the wastewater was 1050 kg.

[0099] Process Optimization 4

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

[0101] Table 3 Solvent selection

[0102] Solvent selection The extracted substances n-Decane A lot of impurities n-Heptane A lot of impurities n-Hexane No product, but the amount of extracted impurities is less than n-heptane n-Pentane No product, but the amount of extracted impurities is less than that of n-heptane Methyl tert-butyl ether A lot of impurities, a small amount of product was extracted Ethyl acetate A lot of impurities, some products are extracted Dichloromethane A lot of impurities, more products are extracted

[0103] When using different solvents to extract small polar impurities, n-heptane and n-decane are the best for removing impurities.

[0104] Process Optimization 5

[0105] Referring to the purification method provided in Example 1, the crude long-acting side chain product was used to prepare the long-acting side chain refined product 1. The steps of the method were the same as those in Example 1, except that the solvents used to extract 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 Extractable substances Dichloromethane Large quantities of product, virtually free of impurities Chloroform Large quantities of product, virtually free of impurities Ethyl acetate Large amount of product, very little impurities Methyl acetate Large amount of product, very little impurities Methyl tert-butyl ether Smaller amount of product, no impurities

[0108] When the product is extracted with dichloromethane, the product loss can be reduced and the amount of solvent used is less; and while extracting the product, the impurities and the product can be separated as much as possible, thus ensuring the quality of the finished product.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for purifying a long-acting side chain, characterized in that: include: The crude long-acting side chain product is mixed with a mixed solvent and the pH is adjusted to 3.1-3.5; wherein the mixed solvent is a mixture of water and epoxy ether; Then, C7-C10 alkane and methyl chloride are used to extract and the first aqueous phase is collected. Next, the first aqueous phase is mixed with sodium chloride and the pH is adjusted to 2.5-2.8, and then the second aqueous phase and the first oil phase are extracted with the methyl chloride and collected. Then, the pH of the second aqueous phase is adjusted to 2.1-2.4, and the second oil phase is extracted with the methyl chloride and collected; Next, the first oil phase and the second oil phase are combined, dried and concentrated to form a long-acting side chain refined product 1; Then, the long-acting side chain refined product 1 is mixed with a salt-forming substance and stirred to form a salt, and then a crystallization solvent and a seed crystal are added to perform crystallization to form a long-acting side chain refined product 2; Then, the long-acting side chain refined product 2 is mixed with methyl chloride and salt water for desalting and phase separation, and then the third aqueous phase is collected. The third aqueous phase is extracted with methyl chloride and the third oil phase is collected.

2. The method for purifying the long-acting side chain according to claim 1, characterized in that: The epoxy ether is selected from tetrahydrofuran, 2-methyltetrahydrofuran and 1,4-dioxane; 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.

3. The method for purifying the long-acting side chain according to claim 1, characterized in that: The C7-C10 alkane is selected from n-heptane and n-decane; Preferably, the methyl chloride is selected from dichloromethane or chloroform.

4. The method for purifying the long-acting side chain according to claim 1, characterized in that: The C7-C10 alkane and the methyl chloride are extracted 1-3 times respectively.

5. The method for purifying the long-acting side chain according to claim 1, characterized in that: For every kilogram of the crude long-acting side chain product, 0.3-0.4 kilogram of sodium chloride is added.

6. The method for purifying the long-acting side chain according to claim 1, characterized in that: The salt-forming substance is selected from barium hydroxide and calcium hydroxide; Preferably, the crystallization solvent is selected from C7-C10 alkanes, more preferably n-heptane and n-decane.

7. The method for purifying the long-acting side chain according to claim 6, characterized in that: The step of forming the long-acting side chain refined product 2 includes: stirring the long-acting side chain refined product 1 and the salt-forming substance at 10-30° C. for 1-2 hours, then adding the C7-C10 alkane and seed crystals and stirring until solid precipitates, and then adding C7-C10 alkane and continuing stirring for 2-3 hours.

8. The method for purifying a long-acting side chain according to claim 1, characterized in that: The salt water comprises concentrated hydrochloric acid, water and sodium chloride; Preferably, the concentration of the salt water is 0.8-1.2%.

9. The method for purifying a long-acting side chain according to claim 1, characterized in that: The long-acting side chain crude products include semaglutide side chain crude products and telpotide side chain crude products.

10. A method for preparing 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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