Process for the one-step synthesis of the key intermediate of the fluoroquinolone antibiotics (s)-7-amino-5-azaspiro[2.4]heptane by transaminases

Abstract

This invention discloses a key intermediate for the one-step synthesis of transaminase into quinolone antibiotics ( S This invention relates to a method for synthesizing 5-azaspiro[2,4]heptane-7-one, belonging to the field of biosynthesis technology for pharmaceutical intermediates. Using 5-azaspiro[2,4]heptane-7-one as a raw material, transaminase as a catalyst, and pyridoxal phosphate as a coenzyme, a one-step asymmetric reductive amination reaction is carried out under the action of an amino donor and a buffer solution. After the reaction, the product is extracted and purified to obtain a key intermediate for quinolone antibiotics ( ). S )-7-amino-5-azaspiro[2,4]heptane. Compared with the prior art, the method provided by this invention solves the technical problems of existing processes requiring two-step reactions of benzyl protection / deprotection, which are cumbersome, costly, and have long production cycles. It achieves efficient, low-cost, and high-purity synthesis of the target intermediate, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis technology of pharmaceutical intermediates, and in particular to a key intermediate for the one-step synthesis of quinolone antibiotics using transaminase. S Method for 7-amino-5-azaspiro[2.4]heptane. Background Technology

[0002] quinolone antibiotics are a class of broad-spectrum quinolone antibacterial drugs that are widely used in clinical anti-infective therapy. S )-7-amino-5-azaspiro[2.4]heptane is the core intermediate for the synthesis of this type of antibiotic, and the quality of its synthesis process directly affects the production cost, production efficiency and product quality of quinolone antibiotics.

[0003] Currently, transaminase catalytic synthesis has been carried out in existing technologies. S Research on 5-7-amino-5-azaspiro[2.4]heptane has shown that the technical route uses 5-benzyl-5-azaspiro[2.4]hept-7-one as the starting material to prepare the target intermediate through a transaminase-catalyzed reaction. However, this synthetic route has significant drawbacks: it requires two additional steps of benzyl protection and deprotection, resulting in cumbersome synthesis steps and a prolonged production cycle. This not only increases the operational complexity of the reaction process but also increases the consumption of raw materials and the generation of wastewater and waste residue. Furthermore, the use of benzyl protecting agents is costly, and the deprotection process requires additional reagents and equipment, further increasing production costs and hindering large-scale industrial production.

[0004] Furthermore, in existing processes, the benzyl protection / deprotection step can lead to a decrease in the optical purity of the target product, the generation of byproducts, a reduction in product yield, and an increase in the difficulty and cost of subsequent separation and purification. Therefore, it is necessary to develop a process that simplifies the steps, reduces costs, and maintains stable yield and optical purity. S The synthesis method of 7-amino-5-azaspiro[2.4]heptane has become a technical problem that urgently needs to be solved in the field of intermediate synthesis of quinolone antibiotics. Summary of the Invention

[0005] The purpose of this invention is to provide a key intermediate for the one-step synthesis of transaminase into quinolone antibiotics. S The method for synthesizing 7-amino-5-azaspiro[2.4]heptane involves using transaminase as a catalyst and pyridoxal coenzyme (PLP) to catalyze an asymmetric reductive amination reaction of the carbonyl group of the starting material 5-azaspiro[2.4]heptane-7-one, thereby synthesizing ( SThe synthesis of 7-amino-5-azaspiro[2.4]heptane solves the technical problems of existing processes that require two-step reactions of benzyl protection / deprotection, which are cumbersome, costly, and time-consuming. It achieves efficient, low-cost, and high-purity synthesis of the target intermediate, making it suitable for large-scale industrial production.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a key intermediate for the one-step synthesis of transaminase into quinolone antibiotics ( S A method for processing 7-amino-5-azaspiro[2,4]heptane includes the following steps: Using 5-azaspiro[2,4]heptan-7-one as the raw material, transaminase as the catalyst, and pyridoxal phosphate as the coenzyme, a one-step asymmetric reductive amination reaction was carried out under the action of an amino donor and a buffer solution. After the reaction, the product was extracted and purified to obtain a key intermediate of quinolone antibiotics. S )-7-amino-5-azaspiro[2,4]heptane.

[0007] Optionally, the molar ratio of the 5-azaspiro[2.4]heptane-7-one, the transaminase, the pyridoxal phosphate and the amino donor is 1:(1.2-2.0):(0.01-0.05):(0.005-0.02).

[0008] Optionally, the transaminase is derived from a recombinant transaminase expressed in Escherichia coli, with the amino acid sequence shown in SEQ ID NO. 1.

[0009] Optionally, the amino donor includes any one of L-alanine, L-glutamic acid, L-aspartic acid, and L-leucine. More preferably, it is L-alanine or L-glutamic acid. Even more preferably, it is L-alanine, which has high amino transfer efficiency, easy separation of byproducts, and low cost, thus reducing raw material costs for industrial production.

[0010] Optionally, the buffer solution includes any one of phosphate buffer, Tris-HCl buffer, citrate-sodium citrate buffer, and borate-borax buffer. Phosphate buffer is more preferred. The concentration of the buffer solution is 0.05-0.2 mol / L. A 0.1 mol / L phosphate buffer is preferred due to its strong buffering capacity, ability to stabilize the pH of the reaction system, and protection of the catalytic activity of the transaminase.

[0011] Optionally, the conditions for the one-step asymmetric reductive amination reaction are: stirring for 8-16 hours under a constant temperature water bath at 25-40℃, and the pH of the reaction system is 6.5-8.5.

[0012] Preferably, the extraction and purification includes the following steps: Add an organic solvent to the system after the reaction is complete, extract 2-3 times, combine the upper organic phases collected after extraction, and then dry them to obtain a clear organic liquid. Distill the clear organic liquid under reduced pressure to obtain the crude product. The crude product was separated by silica gel column chromatography, the target component was collected, and after vacuum distillation, the white solid product was collected as ( ). S )-7-amino-5-azaspiro[2,4]heptane.

[0013] Preferably, the organic solvent includes any one or more of ethyl acetate, dichloromethane, diethyl ether, methyl tert-butyl ether, and butyl acetate. Ethyl acetate is more preferred. Ethyl acetate is even more preferred because it has good solubility for the target product, clear phase separation with the aqueous phase, high extraction efficiency, low toxicity, and is easy to recover, meeting the requirements of green production.

[0014] Preferably, the eluent for the silica gel column chromatography is a mixture of petroleum ether, ethyl acetate, and triethylamine, wherein the volume ratio of petroleum ether, ethyl acetate, and triethylamine is 10:5:1.

[0015] Optionally, the target component is identified by high-performance liquid chromatography (HPLC), with the following identification conditions: C18 column; mobile phase of methanol and water in a volume ratio of 30:70; detection wavelength of 220 nm; and flow rate of 1.0 mL / min.

[0016] The reaction principle of this invention is as follows: With the assistance of coenzyme PLP, transaminase catalyzes an asymmetric reductive amination reaction of the carbonyl group of the starting material 5-azaspiro[2,4]heptane-7-one, transferring the amino group from the amino donor to the carbonyl carbon, directly generating a ( ) with a specific configuration. S The synthesis of 5-azaspiro[2,4]heptane is achieved in a single step without the need for benzyl protection of the nitrogen atom in the raw material or subsequent deprotection steps. Compared with existing processes, the technical solution of this invention solves the technical problems of easy side reactions and low conversion rate in the direct catalytic amination of 5-azaspiro[2,4]heptane-7-one by optimizing the catalyst type and reaction conditions, ensuring the high efficiency of the one-step reaction.

[0017] The present invention discloses the following technical effects: The present invention provides a one-step synthesis method for transaminase catalysis, which, based on the highly specific catalytic activity of a specific transaminase and combined with optimized reaction conditions, achieves (…). S The one-step, highly efficient synthesis of 7-amino-5-azaspiro[2,4]heptane has the following significant advantages compared to existing processes that require two steps of benzyl protection / deprotection: (1) Significantly shortened synthesis steps: The cumbersome two-step reaction of benzyl protection and deprotection in the existing process is eliminated. The target product is directly generated by one-step catalysis using 5-azaspiro[2.4]heptane-7-one as raw material. The synthesis steps are shortened from the original 3 steps (protection-catalysis-deprotection) to 1 step of catalysis + separation and purification. The production cycle is shortened by more than 40%, which greatly improves the production efficiency and reduces the complexity of operation in the production process.

[0018] (2) Significantly reduce production costs: No additional raw materials such as benzyl protecting agents and deprotection reagents are required, which reduces raw material consumption. At the same time, the simplified steps reduce the investment in reaction equipment, reduce energy consumption, and reduce the amount of wastewater and waste residue generated by more than 30%. The difficulty of subsequent separation and purification is reduced, which further reduces production costs. According to calculations, the unit product production cost can be reduced by 25-35%, which significantly enhances the market competitiveness of the product.

[0019] (3) Improved product quality and yield: Avoided side reactions that may occur in the benzyl protection / deprotection steps, reduced by-product formation, and achieved a target product yield of over 92%, with optical purity ( ee The yield (value) is ≥99%, which is higher than that of existing processes (yield of around 85%). ee With a purity of around 98%, it can be directly used in the synthesis of quinolone antibiotics without further purification, reducing the cost and difficulty of subsequent production.

[0020] (4) Green and environmentally friendly, suitable for industrial production: The reaction conditions are mild (25-40℃, normal temperature and pressure), no high temperature and high pressure equipment is required, and energy consumption is low; the selected solvents and buffer solutions are low in toxicity and easy to recycle, and the amount of wastewater and waste residue discharged is small, which meets the requirements of green chemical production; at the same time, the steps are simplified and the operation is simple, making it easy to realize large-scale continuous industrial production, which solves the problem that existing processes are difficult to apply on a large scale. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This invention provides a key intermediate for the one-step synthesis of transaminase into quinolone antibiotics. S Process flow diagram of )-7-amino-5-azaspiro[2.4]heptane; Figure 2 The synthesis of key intermediates for quinolone antibiotics using existing processes (benzyl protection / deprotection) (A) and the process of this invention (B) SComparison of steps for 7-amino-5-azaspiro[2.4]heptane. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] This invention uses 5-azaspiro[2,4]heptan-7-one as the starting material, transaminase as the catalyst, and pyridoxal phosphate (PLP) as the coenzyme. Under the action of an amino donor and a buffer solution, a one-step asymmetric reductive amination reaction is carried out under certain temperature and pH conditions to directly generate a key intermediate of quinolone antibiotics. S )-7-amino-5-azaspiro[2,4]heptane, after the reaction, was purified to obtain the high-purity target product without the need for any introduction or removal of protecting groups. The synthetic process is as follows: Figure 1 .

[0029] The above solution will be further illustrated below with specific embodiments.

[0030] Example 1: One-step synthesis of 5-azaspiro[2,4]heptane-7-one catalyzed by transaminase ( S )-7-amino-5-azaspiro[2,4]heptane The specific operating steps include: (1) Preparation of the reaction system In a 500 mL three-necked flask, add 200 mL of 0.1 mol / L phosphate buffer (pH=7.5), then add 10 mmol of 5-azaspiro[2.4]heptane-7-one, 15 mmol of L-alanine (amino donor), 0.3 mmol of recombinant transaminase (this enzyme was directionally modified (the original GenBank accession number was MH196528, obtained by expression in E. coli; the modification method and enzyme sequence have been disclosed by the applicant in the authorized patent "CN201811449183.X A mutant ω-transaminase that can catalyze the five-membered ring intermediate of sitafloxacin," see the ω-transaminase mutant in that patent for details), and 0.1 mmol of coenzyme PLP. Stir the mixture thoroughly with a magnetic stirrer, and adjust the pH of the system to 7.5 using dilute hydrochloric acid or sodium hydroxide solution to obtain the reaction mixture.

[0031] (2) One-step catalytic reaction A three-necked flask was placed in a 30°C constant temperature water bath, and the magnetic stirring speed was maintained at 300 r / min to carry out the catalytic reaction. Samples were taken every 2 hours during the reaction, and the reaction progress was monitored by high performance liquid chromatography (HPLC). The HPLC detection conditions were: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase methanol:water = 30:70 (v / v), detection wavelength 220 nm, flow rate 1.0 mL / min, and column temperature 30°C. After 12 hours of reaction, the conversion rate of 5-azaspiro[2.4]heptane-7-one was detected to be 98.5%, and the reaction was stopped.

[0032] (3) Separation and purification Add 100 mL of ethyl acetate to the reaction termination solution, stir and extract for 15 min, allow to stand and separate into layers, take the upper organic phase; extract the lower aqueous phase twice more with 100 mL of ethyl acetate, combine the three organic phases, add 10 g of anhydrous sodium sulfate and dry for 2 h, filter to remove anhydrous sodium sulfate, and obtain a clear organic liquid; place the organic liquid in a rotary evaporator and remove ethyl acetate by vacuum distillation at 50 °C and 0.08 MPa to obtain the crude product.

[0033] The crude product was separated by silica gel column chromatography using petroleum ether:ethyl acetate:triethylamine = 10:5:1 (v / v / v). The target component was collected (confirmed by HPLC), and the eluent was removed again by vacuum distillation to obtain a white solid product (S)-7-amino-5-azaspiro[2,4]heptane, weighing 1.18 g, with a yield of 93.6%. The product was detected by chiral high-performance liquid chromatography. ee The value is 99.2%.

[0034] Example 2: One-step synthesis of 5-azaspiro[2,4]heptane-7-one catalyzed by transaminase ( S )-7-amino-5-azaspiro[2,4]heptane The specific operating steps include: (1) Preparation of the reaction system In a 500 mL three-necked flask, add 200 mL of 0.05 mol / L phosphate buffer (pH=6.5), then add 10 mmol of 5-azaspiro[2.4]heptane-7-one, 12 mmol of L-alanine (amino donor), 0.1 mmol of recombinant transaminase, and 0.05 mmol of coenzyme PLP. Stir the mixture thoroughly with a magnetic stirrer and adjust the pH of the system to 6.5 using dilute hydrochloric acid or sodium hydroxide solution to obtain the reaction mixture.

[0035] (2) One-step catalytic reaction A three-necked flask was placed in a constant temperature water bath at 25℃, and the magnetic stirring speed was maintained at 300 r / min to carry out the catalytic reaction. Samples were taken every 2 hours during the reaction, and the reaction progress was monitored by high performance liquid chromatography (HPLC). The HPLC detection conditions were as follows: column C18 (250 mm × 4.6 mm, 5 μm), mobile phase methanol:water = 30:70 (v / v), detection wavelength 220 nm, flow rate 1.0 mL / min, and column temperature 30℃. After 8 hours of reaction, the conversion rate of 5-azaspiro[2.4]heptane-7-one was detected to be 98.0%, and the reaction was stopped.

[0036] (3) Same as (3) in Example 1.

[0037] The final white solid product (S)-7-amino-5-azaspiro[2,4]heptane was obtained, with a mass of 1.16 g and a yield of 92.1%. The product was detected by chiral high-performance liquid chromatography. ee The value is 99.0%.

[0038] Example 3: One-step synthesis of 5-azaspiro[2,4]heptane-7-one catalyzed by transaminase ( S )-7-amino-5-azaspiro[2,4]heptane The specific operating steps include: (1) Preparation of the reaction system In a 500 mL three-necked flask, add 200 mL of 0.1 mol / L phosphate buffer (pH=8.5), then add 10 mmol of 5-azaspiro[2.4]heptane-7-one, 20 mmol of L-alanine (amino donor), 0.5 mmol of recombinant transaminase, and 0.2 mmol of coenzyme PLP. Stir the mixture thoroughly with a magnetic stirrer and adjust the pH of the system to 8.5 using dilute hydrochloric acid or sodium hydroxide solution to obtain the reaction mixture.

[0039] (2) One-step catalytic reaction A three-necked flask was placed in a 40°C constant temperature water bath, and the magnetic stirring speed was maintained at 300 r / min to carry out the catalytic reaction. Samples were taken every 2 hours during the reaction, and the reaction progress was monitored by high performance liquid chromatography (HPLC). The HPLC detection conditions were: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase methanol:water = 30:70 (v / v), detection wavelength 220 nm, flow rate 1.0 mL / min, and column temperature 30°C. After 8 hours of reaction, the conversion rate of 5-azaspiro[2.4]heptane-7-one was detected to be 95.5%, and the reaction was stopped.

[0040] (3) Same as (3) in Example 1.

[0041] The final white solid product (S)-7-amino-5-azaspiro[2,4]heptane was obtained, with a mass of 1.17 g and a yield of 93.0%. The product was detected by chiral high-performance liquid chromatography. ee The value is 99.1%.

[0042] Example 4: Effect of different amino donors on the reaction effect Using the same reaction conditions and operating procedures as in Example 1, except that L-alanine was replaced with an equimolar amount of L-glutamic acid, while all other conditions remained unchanged. After 14 h of reaction, the conversion rate of 5-azaspiro[2,4]heptane-7-one was 96.8%, yielding ( S The yield of 7-amino-5-azaspiro[2,4]heptane was 90.2%. ee The value is 99.0%.

[0043] The results showed that, compared with L-glutamic acid, L-alanine had higher reaction efficiency and slightly higher product yield when used as an amino donor. Relatively speaking, L-alanine had lower cost and was more suitable for industrial production.

[0044] Comparative Example 1: Effect of different transaminases on the reaction effect Using the same reaction conditions and operating procedures as in Example 1, except that the recombinant transaminase was replaced with a common commercial transaminase (derived from pig heart; Sigma-Aldrich; catalog number: T7684; specification: ≥5 units / mg), all other conditions remained unchanged. After 16 h of reaction, the conversion rate of 5-azaspiro[2,4]heptane-7-one was 8.3%, yielding ( S The yield of 7-amino-5-azaspiro[2,4]heptane was 2.1%. ee The value is 77.5%.

[0045] Compared with Comparative Example 1, it can be seen that the recombinant transaminase selected by the present invention, which has been directionally modified, has higher catalytic activity and specificity for 5-azaspiro[2.4]heptane-7-one, which can significantly improve the reaction conversion rate, product yield and optical purity.

[0046] Comparative Example 2: Synthesis using existing benzyl protection / deprotection processes ( S )-7-amino-5-azaspiro[2,4]heptane Starting with 5-benzyl-5-azaspiro[2.4]hepta-7-one, the following steps were performed: First, a benzyl protection reaction was carried out: 5-benzyl-5-azaspiro[2.4]hepta-7-one and benzyl chloride were mixed at a molar ratio of 1:1.2 and reacted under alkaline conditions for 4 h to obtain the protected product. Second, a transaminase-catalyzed reaction was carried out: the protected product, transaminase, coenzyme PLP, and amino donor were added to a buffer solution and reacted at 30 °C for 12 h. Third, a deprotection reaction was carried out: palladium catalyst on carbon was added to the reaction solution, hydrogen gas was introduced, and the reaction was carried out at room temperature and pressure for 6 h to remove the benzyl group. Finally, the target product was obtained by separation and purification, with a total yield of 84.7%. ee The value was 98.1%, the production cycle was 26 hours, and the unit product production cost was 32% higher than that of Example 1.

[0047] Comparing Example 1 and Comparative Example 2 (see the comparison chart of synthesis processes) Figure 2 As can be seen, the one-step synthesis method of the present invention is superior to the existing process in terms of yield and optical purity, significantly shortens the production cycle (46%), and significantly reduces the production cost, showing obvious advantages.

[0048] The amino acid sequence (SEQ ID NO.1) of the recombinant transaminase of the present invention is as follows: MEDQKEQWIFLNDELVKKEDAKISVYDHGFLWGDGVFEGIRVYNGNIFRMKEHLDRLYDSARSIMLNIPYSLEELTEKMIHTVERNGLKDAYIRLVVSRGAGDLGLDPNNCGRANTVIIVEPLAIFPKHLYETGIDIVTVPTRRNRPDVLSP KVKSLNFLNNILVRIEAHMAGVSEALMLNDQGYVAEGSADNVFIYKKGKLYTPPGYIGALEGMTRNAIMEIAEDLGYEVKEEPFTRHDVYTAEEVFLTGAAAEVIAVVKVDGRMIGEGKPGFHTNKLLEQFRKRVVEEGEKVVFTDENIHAS.

[0049] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A key intermediate for one-step synthesis of quinolone antibiotics from a transaminase ( S The method for using 7-amino-5-azaspiro[2,4]heptane is characterized by, Includes the following steps: Using 5-azaspiro[2,4]heptan-7-one as the raw material, transaminase as the catalyst, and pyridoxal phosphate as the coenzyme, a one-step asymmetric reductive amination reaction was carried out under the action of an amino donor and a buffer solution. After the reaction, the product was extracted and purified to obtain a key intermediate of quinolone antibiotics. S )-7-amino-5-azaspiro[2,4]heptane.

2. The method as described in claim 1, characterized in that, The molar ratio of the 5-azaspiro[2.4]heptane-7-one, the transaminase, the pyridoxal phosphate, and the amino donor is 1:(1.2-2.0):(0.01-0.05):(0.005-0.02).

3. The method as described in claim 1, characterized in that, The transaminase is derived from a recombinant transaminase expressed in Escherichia coli, and its amino acid sequence is shown in SEQ ID NO.

1.

4. The method as described in claim 1, characterized in that, The amino donor includes any one of L-alanine, L-glutamic acid, L-aspartic acid, and L-leucine.

5. The method as described in claim 1, characterized in that, The buffer solution includes any one of phosphate buffer, Tris-HCl buffer, citrate-sodium citrate buffer, and borate-borax buffer, and the concentration of the buffer solution is 0.05-0.2 mol / L.

6. The method as described in claim 1, characterized in that, The conditions for the one-step asymmetric reductive amination reaction are: stirring for 8-16 hours in a constant temperature water bath at 25-40℃, with a pH of 6.5-8.5 in the reaction system.

7. The method as described in claim 1, characterized in that, The extraction and purification process includes the following steps: Add an organic solvent to the system after the reaction is complete, extract 2-3 times, combine the upper organic phases collected after extraction, and then dry them to obtain a clear organic liquid. Distill the clear organic liquid under reduced pressure to obtain the crude product. The crude product was separated by silica gel column chromatography, the target component was collected, and after vacuum distillation, the white solid product was collected as ( ). S )-7-amino-5-azaspiro[2,4]heptane.

8. The method as described in claim 7, characterized in that, The organic solvent includes any one or more of ethyl acetate, methyl tert-butyl ether, and butyl acetate.

9. The method as described in claim 7, characterized in that, The eluent for the silica gel column chromatography is a mixture of petroleum ether, ethyl acetate, and triethylamine, wherein the volume ratio of petroleum ether, ethyl acetate, and triethylamine is 10:5:

1.

10. The method as described in claim 7, characterized in that, The target component was identified by high-performance liquid chromatography (HPLC) under the following conditions: C18 column; mobile phase of methanol and water in a volume ratio of 30:70; detection wavelength of 220 nm; and flow rate of 1.0 mL / min.

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