Purification method and preparation method of fatty acid side chain

Abstract

The invention relates to the technical field of purification and refining of compounds, in particular to a purification method and a preparation method of a fatty acid side chain. The purification method of the fatty acid side chain comprises the following steps: mixing a fatty acid side chain crude product with a mixed solvent, and adjusting the pH value to 2.7-3.4; then, C6-C10 alkane and chloromethane are sequentially used for extraction, and a first water phase is collected; mixing the first water phase with chloride, adjusting the pH value to 2.3-2.6, extracting with chloromethane, and collecting a second water phase and a first oil phase; then adjusting the pH value of the second water phase to 2.0-2.2, extracting with chloromethane, and collecting a second oil phase; mixing the first oil phase and the second oil phase, washing with acid salt water, and then drying and concentrating. According to the purification method, the output of three wastes such as waste organic solvents and solid wastes is reduced, the production period is also greatly shortened, the production time is saved, the production cost is saved, and favorable conditions are provided for large-scale production of products.

Description

Technical Field

[0001] The present invention relates to the technical field of compound purification and refining, and in particular to a method for purifying fatty acid side chains and a method for preparing the same. Background Art

[0002] Insulin is a special polypeptide hormone compound for the treatment of diabetes. However, since insulin itself is a protein, it is easily hydrolyzed and inactivated by proteases in the stomach and intestines. Therefore, it can only be used by injection. In order to relieve the pain of injection for patients, the long-acting drug is an ideal means. For example, semaglutide developed by Novo Nordisk and telpotide developed by Eli Lilly and Company, the lysine in the peptide chain is modified by connecting short-chain PEG and C18 or C20 diacid fatty chains, so that it can mediate and promote the strong binding of semaglutide and telpotide to albumin and reduce renal clearance. Compared with liraglutide with C16 side chain, the affinity of semaglutide and telpotide side chains for albumin is enhanced by 5 to 6 times. Binding with albumin can increase the molecular weight of the product, increase its molecular size, and it can also carry a large number of water molecules. Secondly, PEG modification makes previously insoluble proteins not only easy to dissolve, but also highly mobile. In addition, PEG modification can reduce the filtration of drugs by the kidneys, reduce its pyrogenicity, and reduce its digestion by proteases, improving its delivery by protecting the molecule from the attack of the human immune system. At the same time, because it escapes the body's defense mechanism, it stays at the site of action much longer, avoiding rapid clearance by the kidneys and preventing metabolic degradation, prolonging the half-life in the body, and achieving a long circulation effect.

[0003] Fatty acid side chains are key intermediates for the synthesis of long-acting side fragments of diabetes drugs (GLP-1). The above fatty acid side chains are liquid or paste at room temperature. The current purification process is mainly based on column chromatography, which produces a huge amount of three wastes. The special requirements of chromatography equipment not only limit its single batch production, but also increase the normal production cycle, bringing huge challenges to the large-scale production of products.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a method for purifying fatty acid side chains and a method for preparing the same. The purification method provided in the embodiment of the present invention reduces the generation of three wastes, such as waste organic solvents and solid wastes, and also greatly shortens the production cycle, saves production time, saves production costs, and provides favorable conditions for large-scale production of products.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a method for purifying fatty acid side chains, comprising: mixing crude fatty acid side chains with a mixed solvent and adjusting the pH to 2.7-3.4; wherein the mixed solvent is a mixture of water and epoxy ether;

[0008] Then, C6-C10 alkane and methyl chloride are used to extract and the first aqueous phase is collected;

[0009] Next, the first aqueous phase is mixed with chloride and the pH is adjusted to 2.3-2.6, and then the methyl chloride is used for extraction and the second aqueous phase and the first oil phase are collected;

[0010] Then, the pH of the second aqueous phase is adjusted to 2.0-2.2, and then the second oil phase is extracted with the methyl chloride and collected;

[0011] Next, the first oil phase and the second oil phase are mixed and washed with an aqueous acid salt solution, followed by drying and concentration.

[0012] In an alternative embodiment, the epoxy ether is selected from tetrahydrofuran, 2-methyltetrahydrofuran and 1,4-dioxane.

[0013] In an optional embodiment, 3-4 kg of water and 0.2-0.3 kg of the epoxy ether are added per kg of the crude fatty acid side chain product.

[0014] In an optional embodiment, the C6-C10 alkane is selected from n-heptane and n-decane;

[0015] Preferably, the methyl chloride is selected from dichloromethane.

[0016] In an optional embodiment, each time the C6-C10 alkane is used for extraction, 0.5-0.7 kg of the C6-C10 alkane is added per kg of the crude fatty acid side chain product;

[0017] Preferably, each time the methyl chloride is used for extraction, 3-4 kg of the methyl chloride is added for every kg of the crude fatty acid side chain product.

[0018] In an optional embodiment, the pH of the salt water is 2.6-2.8.

[0019] In an optional embodiment, the step of preparing the salt water comprises: mixing chloride and water, and then adjusting the pH to 2.6-2.8 using hydrochloric acid.

[0020] In an alternative embodiment, drying comprises drying using anhydrous magnesium sulfate.

[0021] In an optional embodiment, the crude fatty acid side chain includes a crude semaglutide side chain not linked to Lys and a crude tepoxetine side chain not linked to Lys.

[0022] In a second aspect, the present invention provides a method for preparing fatty acid side chains, comprising: the method for purifying fatty acid side chains described in any one of the aforementioned embodiments.

[0023] The present invention has the following beneficial effects: The embodiment of the present invention provides a new method for purifying fatty acid side chains, which can separate impurities in fatty acid side chains and improve the purity of fatty acid side chains only by adjusting pH and extraction, which not only shortens the production cycle, but also timely reduces the generation of waste organic solvents, solid waste and other three wastes. It saves production costs and provides favorable conditions for large-scale production of products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is a high-resolution spectrum of the side chain of semaglutide provided in Example 1 of the present invention;

[0026] Figure 2 This is a high-performance liquid chromatography (HPLC) spectrum of the side chain of telpotide provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0027] 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.

[0028] The fatty acid side chain is a crude semaglutide side chain not linked to Lys and a telpotide side chain not linked to Lys, specifically, selected from the compounds represented by the following structural formula:

[0029]

[0030] The fatty acid side chain can be used to prepare semaglutide or telportide. In the prior art, the purification method of the fatty acid side chain generally adopts column chromatography. Although column chromatography can make the purity of the fatty acid side chain reach 99%, when column chromatography is used for purification, the operation is complicated, the production cycle is very long, and the amount of waste solvent, waste water and solid waste generated is significantly reduced, which further saves production costs.

[0031] The impurities it contains include 3AEEA impurity, single AEEA impurity and de-tert-butyl impurity. The structural formula of the specific impurities is as follows:

[0032]

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

[0034] Specifically, the purification method of the above fatty acid side chains is as follows:

[0035] The crude fatty acid side chain product is mixed with a mixed solvent, wherein the crude fatty acid side chain product is prepared according to patent CN115368234A. 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 to each kilogram of the crude fatty acid side chain product. The epoxy ether is selected from tetrahydrofuran, 2-methyltetrahydrofuran and 1,4-dioxane.

[0036] The use of the mixed solvent can dissolve fatty acid side chains and impurities, which is beneficial for subsequent separation.

[0037] Then, the pH is adjusted to 2.7-3.4 with acid, such as any value between 2.7-3.4 or a range between any two values ​​such as 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3 and 3.4, for example, preferably 2.8-3.2. The pH is adjusted with an acidic substance such as hydrochloric acid.

[0038] Then, the solution is extracted with C6-C10 alkane to collect the aqueous phase to remove epoxy ether and small polar impurities. The C6-C10 alkane is selected from n-heptane and n-heptane. Each time the C6-C10 alkane is used for extraction, 0.5-0.7 kg of the C6-C10 alkane is added for each kg of the crude fatty acid side chain.

[0039] The aqueous phase is further extracted with methyl chloride and the first aqueous phase is collected, wherein the methyl chloride is selected from dichloromethane, and 3-4 kg of methyl chloride is added for each kg of the fatty acid side chain during each extraction.

[0040] The first aqueous phase is mixed with chloride and the pH is adjusted to 2.3-2.6. Specifically, the first aqueous phase and chloride are stirred, and then the pH is adjusted to 2.3-2.6 with acid. The above-mentioned chloride includes but is not limited to sodium chloride. 0.3-0.4 kg of chloride is added for each kg of crude fatty acid side chain.

[0041] Chloride is used to reduce the solubility of fatty acid side chains in water and to improve the efficiency of extracting fatty acid side chains with methyl chloride.

[0042] Then, methyl chloride is added to the above solution for extraction and the second water phase and the first oil phase are collected respectively. The methyl chloride is dichloromethane, and 3-4 kg of methyl chloride is added for each kg of crude fatty acid side chain product during each extraction.

[0043] Next, the pH of the second aqueous phase is adjusted to 2.0-2.2, methyl chloride is added to the above solution for extraction, and the second oil phase is collected. The methyl chloride is dichloromethane, and 3-4 kg of methyl chloride is added for each kilogram of crude fatty acid side chain product during each extraction.

[0044] Then, the first oil phase and the second oil phase are combined, that is, the methyl chloride phase obtained by extraction at pH = 2.3-2.6 and 2.0-2.2 is combined, and then washed with a salt solution. Specifically, the methyl chloride phase is washed multiple times with a salt solution at pH = 2.6-2.8, wherein the preparation step of the salt solution comprises mixing chloride and water, and then adjusting the pH to 2.6-2.8 with hydrochloric acid.

[0045] After washing, the product is dried with anhydrous magnesium sulfate, filtered and concentrated to obtain the desired fatty acid side chain.

[0046] In a second aspect, the present invention provides a method for preparing fatty acid side chains, comprising: the method for purifying fatty acid side chains described in any one of the aforementioned embodiments.

[0047] It should be noted that the purity detection method provided in the embodiment of the present invention is as follows:

[0048] Chromatographic column: Phenomenex Lux i-cellulose-5 (4.6*250mm, 3μm);

[0049] Mobile phase: 0.1% glacial acetic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B;

[0050] Injection volume: 10 μl;

[0051] Column temperature: 35 °C;

[0052] Flow rate: 1ml / min;

[0053] Detection wavelength: 210nm;

[0054] Gradient elution method:

[0055]

[0056] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0057] Example 1

[0058] A method for purifying fatty acid side chains according to an embodiment of the present invention comprises:

[0059] 15 kg of crude semaglutide side chain (prepared with reference to CN115368234A) (the crude product has a purity of 88%) (the crude product is not connected to Lys) was added with 50 kg of water and 4 kg of tetrahydrofuran, stirred evenly, and the pH was adjusted to 2.8 with 4 mol / L hydrochloric acid (precision control with a pH meter).

[0060] Add n-heptane to extract twice (2*10kg) and collect the aqueous phase to remove tetrahydrofuran and small polar impurities.

[0061] Dichloromethane was added to the aqueous phase to extract impurities twice (2*45kg).

[0062] After the impurities are extracted, 5 kg of sodium chloride is first added to the aqueous phase; the mixture is stirred and the pH is adjusted to 2.3 with 4 mol / L hydrochloric acid (precisely controlled by a pH meter); dichloromethane is added and extracted twice (2*45 kg).

[0063] The aqueous phase after extraction was adjusted to pH = 2.0 with 4 mol / L hydrochloric acid (precisely controlled by a pH meter), and dichloromethane was added for extraction once (1*45 kg).

[0064] Combine the dichloromethane phases extracted three times at pH = 2.3 and 2.0, and wash the dichloromethane phase twice with acid salt water at pH = 2.6 (2*10kg). The acid salt water preparation step includes: mixing 20kg of sodium chloride and 80kg of water, controlling the temperature at 15-25°C, and adjusting the pH to 2.6 with 6mol / L hydrochloric acid.

[0065] Then, anhydrous magnesium sulfate was added for drying, filtration and concentration were performed to obtain the finished product of semaglutide side chain.

[0066] 10.0 kg of the semaglutide side chain was detected with a purity of 99.734%. The high-performance liquid chromatography (HPLC) spectrum is shown in Figure 1 The content of single AEEA impurity was 0.038%, 3AEEA impurity and de-tert-butyl impurity were not detected, and the maximum content of unknown impurity was 0.060%.

[0067] Example 2

[0068] A method for purifying fatty acid side chains according to an embodiment of the present invention comprises:

[0069] 15 kg of crude tepoxetine side chain (prepared with reference to CN115368234A) (the crude product has a purity of 89%) (the crude product is not connected to Lys) was added with 50 kg of water and 4 kg of 2-methyltetrahydrofuran, stirred evenly, and the pH was adjusted to 3.2 with 4 mol / L hydrochloric acid (precision control with a pH meter).

[0070] n-heptane was added for extraction twice (2*10 kg) and the aqueous phase was collected to remove 2-methyltetrahydrofuran and small polar impurities.

[0071] Dichloromethane was added to the aqueous phase to extract impurities twice (2*45kg).

[0072] After the impurities are extracted, 5 kg of sodium chloride is first added to the aqueous phase; the mixture is stirred and adjusted to pH = 2.6 with 4 mol / L hydrochloric acid (precisely controlled by a pH meter); dichloromethane is added and extracted twice (2*45 kg).

[0073] The aqueous phase after extraction was adjusted to pH = 2.2 with 4 mol / L hydrochloric acid (precisely controlled by a pH meter), and dichloromethane was added for extraction once (1*45 kg).

[0074] Combine the dichloromethane phases extracted three times at pH = 2.6 and 2.2, and wash the dichloromethane phase twice with acid salt water at pH = 2.8 (2*10kg). The acid salt water preparation step includes: mixing 20kg sodium chloride and 80kg water, controlling the temperature at 15-25°C, and adjusting the pH to 2.8 with 6mol / L hydrochloric acid.

[0075] Then, anhydrous magnesium sulfate was added for drying, filtration and concentration were performed to obtain the finished product of tepoxetine side chain.

[0076] 9.8 kg of the tepoxetine side chain was detected with a purity of 99.478%. Figure 2 The content of single AEEA impurity was 0.014%, 3AEEA impurity and de-tert-butyl impurity were not detected, and the content of the maximum unknown impurity was 0.086%.

[0077] Comparative Example

[0078] The following process was used to purify the crude product of semaglutide side chain, and the specific steps were as follows:

[0079] 180 kg of crude semaglutide side chain (prepared with reference to CN114805543A) (the purity of the crude product is 88%) was dissolved in 900 kg of ethyl acetate, washed with 0.1 mol / L hydrochloric acid 3 times, each time using 60 kg, and the organic phase was collected; the organic phase was washed once with drinking water and 3 times with saturated brine, each time using 130 kg, and the organic phase was collected; 55 kg of anhydrous magnesium sulfate was added to the organic phase for drying, filtering, and concentrating; 100 kg of ester was dissolved at 40-45°C and divided into 4 equal parts. Each part was added to a silica gel chromatography column for loading. Each chromatography column was loaded with 120 kg of silica gel. After loading each chromatography column, impurities were eluted with ethyl acetate-n-heptane (m / m=4:1). The amount used for each chromatography column was 8400 kg. The product was then eluted with dichloromethane-methanol (m / m=20:1). The amount used for each chromatography column was 1900 kg. The solution of the eluted product received was concentrated to obtain 100 kg of the desired product.

[0080] The existing purification process produces 42,500 kg of organic waste liquid, 535 kg of solid waste, and 700 kg of waste water.

[0081] The same amount of crude semaglutide side chain was treated by the method of Example 1, and the method produced 2490 kg of organic waste liquid, 50 kg of solid waste and 950 kg of waste water.

[0082] Process Optimization 1

[0083] Purification was performed according to the purification method for fatty acid side chains provided in Example 1, except that the extraction reagents used for the aqueous phase after adjusting the pH to 2.3 and 2.0 were different, and dichloromethane was replaced with the solvent corresponding to the following table. The extraction results are also shown in the following Table 1.

[0084] Table 1 Extraction solvent selection

[0085]

[0086] It can be seen that if the extraction solvent is changed, the impurity content in the extracted material may increase, or the desired product cannot be extracted at all, which further illustrates that only methyl chloride defined in the embodiment of the present invention can be used for extraction.

[0087] Process Optimization 2

[0088] The first extract was prepared by the purification method of the fatty acid side chain provided in Example 1: 150g of the crude product of the telportin side chain (prepared with reference to CN115368234A) was added with 500g of water and 40g of tetrahydrofuran, stirred evenly, and the pH was adjusted to 2.8 with 4mol / L hydrochloric acid (pH meter for precise control). n-heptane was added to extract twice (2*100g) and the aqueous phase was collected. Dichloromethane was added to the aqueous phase to extract impurities twice (2*450g). Then anhydrous sodium magnesium sulfate was added to dry the oil phase, filtered and concentrated to obtain the first extract of the semaglutide side chain. The crude product was extracted at different pH values, and the first extract obtained by extraction was detected. The specific methods and processes were all referred to Example 1, with the only difference being the different pH values. See Table 2 for details.

[0089] Table 2 pH selection

[0090]

[0091] It can be seen that if the pH value of the extraction is changed, the product content in the material extracted when the 3AEEA impurity is extracted may increase, or the desired product may be completely extracted when the 3AEEA impurity is extracted. According to the above results, when using salt water to wash the impurities of the first extract, if the pH value of the washing is changed, it may result in the failure to remove the single AEEA impurity during washing, resulting in an increase in product impurities, or the desired product may be completely washed away during washing, further indicating that only the pH specified in the embodiment of the present invention can be used for extraction.

[0092] 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 fatty acid side chains, characterized in that: include: The crude fatty acid side chain product is mixed with a mixed solvent and the pH is adjusted to 2.7-3.4; wherein the mixed solvent is a mixture of water and epoxy ether; Then, C6-C10 alkane and methyl chloride are used to extract and the first aqueous phase is collected; Next, the first aqueous phase is mixed with chloride and the pH is adjusted to 2.3-2.6, and then the methyl chloride is used for extraction and the second aqueous phase and the first oil phase are collected; Then, the pH of the second aqueous phase is adjusted to 2.0-2.2, and then the second oil phase is extracted with the methyl chloride and collected; Next, the first oil phase and the second oil phase are mixed and washed with an aqueous acid salt solution, followed by drying and concentration.

2. The method for purifying fatty acid side chains according to claim 1, characterized in that: The epoxy ether is selected from tetrahydrofuran, 2-methyltetrahydrofuran and 1,4-dioxane.

3. The method for purifying fatty acid side chains according to claim 1, characterized in that: For every kilogram of the crude fatty acid side chain product, 3-4 kilograms of water and 0.2-0.3 kilograms of the epoxy ether are added.

4. The method for purifying fatty acid side chains according to claim 1, characterized in that: The C6-C10 alkane is selected from n-heptane and n-decane; Preferably, the methyl chloride is selected from dichloromethane.

5. The method for purifying fatty acid side chains according to claim 1, characterized in that: Each time the C6-C10 alkane is used for extraction, 0.5-0.7 kg of the C6-C10 alkane is added for every kg of the crude fatty acid side chain product; Preferably, each time the methyl chloride is used for extraction, 3-4 kg of the methyl chloride is added for every kg of the crude fatty acid side chain product.

6. The method for purifying fatty acid side chains according to claim 1, characterized in that: The pH of the salt water is 2.6-2.

8.

7. The method for purifying fatty acid side chains according to claim 1, characterized in that: The preparation steps of the salt water include: mixing chloride and water, and then adjusting the pH to 2.6-2.8 with hydrochloric acid.

8. The method for purifying fatty acid side chains according to any one of claims 1 to 7, characterized in that: Drying includes drying using anhydrous magnesium sulfate.

9. The method for purifying fatty acid side chains according to any one of claims 1 to 7, characterized in that: The crude fatty acid side chain includes a crude semaglutide side chain not linked to Lys and a crude telpotide side chain not linked to Lys.

10. A method for preparing a fatty acid side chain, characterized in that: include: A method for purifying fatty acid side chains according to any one of claims 1 to 9.

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