WO2023000516A1

WO2023000516A1 - Purification method for paeoniflorin-6-o'-benzene sulfonate

Info

Publication number: WO2023000516A1
Application number: PCT/CN2021/124535
Authority: WO
Inventors: 许文东; 唐顺之; 袁诚; 刘菊妍; 蔡鸿飞; 白柏; 傅玉萍; 李菁; 李松涛; 彭万才; 李继荣; 魏劭恒; 李佳俐; 满兴战
Original assignee: 广州白云山汉方现代药业有限公司
Priority date: 2021-07-23
Filing date: 2021-10-19
Publication date: 2023-01-26
Also published as: CN115677792A

Abstract

The present invention provides a purification method for paeoniflorin-6-O'-benzene sulfonate. According to the purification method, the purification process for paeoniflorin-6-O'-benzene sulfonate can be reduced, and impurities, such as benzenesulfonyl chloride and derivatives thereof, iron, and heavy metals, in the prepared paeoniflorin-6-O'-benzene sulfonate crude product can be effectively removed, to obtain paeoniflorin-6-O'-benzene sulfonate of which the content is 98% or more. In addition, under the condition of ensuring high content, the energy consumption is low, the solvent dosage is small, the time consumption is low, and the method is thus suitable for industrial production of paeoniflorin-6-O'-benzene sulfonate.

Description

A kind of purification method of paeoniflorin-6-O'-benzenesulfonate

technical field

The invention belongs to the technical field of compound purification, and in particular relates to a purification method of paeoniflorin-6-O'-benzenesulfonate.

Background technique

Paeoniforin (Paeoniforin, Pae) is derived from plants such as peony root and peony root, with a large content, and is the main component of total paeoniflorin. It has a wide range of pharmacological activities, including anti-inflammatory, immune regulation, antipyretic and analgesic, lowering blood pressure, antispasmodic and anti-tumor, etc. It has low toxicity and few adverse reactions. It has been used in the clinical treatment of rheumatoid arthritis. Although Pae has significant pharmacological effects, its poor oral absorption and slow onset of action limit its use in large doses.

Therefore, Wei Wei et al. conducted a derivatization study on Pae and found that its derivative paeoniflorin-6-O'-benzenesulfonate has better drug absorption in animals, showing slower drug clearance, retention Long time and high bioavailability.

The synthesis of paeoniflorin-6-O'-benzenesulfonate is prepared by the reaction of paeoniflorin and benzenesulfonyl chloride. Since there are multiple hydroxyl groups on paeoniflorin, the reaction is not selective, so more impurities are generated in the reaction. In the prior art, even if paeoniflorin with a content of at least 98% is used as a raw material to prepare paeoniflorin-6-O'-benzenesulfonate, the product is purified by silica gel chromatography column separation. On the one hand, only through silica gel separation Often there will be a small amount of unreacted benzenesulfonyl chloride and some side reaction products whose polarity is close to that of paeoniflorin-6-O'-benzenesulfonate; on the other hand, even if the paeoniflorin-6- O'-benzenesulfonate, but the high content of reaction raw materials leads to limited production of paeoniflorin-6-O'-benzenesulfonate. However, carrying out multiple column chromatography for paeoniflorin-6-O'-benzenesulfonate with a content of 95% to 98% will not only consume a large amount of eluent and silica gel, but also lose a large amount of samples, reducing the amount of paeoniflorin. - Yield of 6-O'-benzenesulfonate.

Contents of the invention

The present invention aims to solve at least one of the technical problems in the above-mentioned prior art.

For this reason, the present invention proposes a kind of purification method of paeoniflorin-6-O'-benzenesulfonate, and this purification method can reduce the purification process of paeoniflorin-6-O'-benzenesulfonate, while ensuring high content , low energy consumption, less solvent consumption, less time-consuming, suitable for industrial production of paeoniflorin-6-O'-benzenesulfonate.

Paeoniflorin contains multiple hydroxyl groups, and the reaction between paeoniflorin and benzenesulfonyl chloride is not selective. As shown in Figure 1, after the reaction, in addition to the main component of paeoniflorin-6-O'-benzenesulfonate, there are many Up to 15 impurities were formed. Among them, impurity 7 is the reaction product of residual paeonifloride glycosides in paeoniflorin and benzenesulfonyl chloride, and the other 14 impurities are impurities generated during the reaction. It can be seen that after the reaction of paeoniflorin-6-O'-benzenesulfonate is completed, the components of the reaction system are relatively complicated, and refining is required to obtain the finished product of paeoniflorin-6-O'-benzenesulfonate with a higher content.

Therefore, the present invention proposes a purification method of paeoniflorin-6-O'-benzenesulfonate, comprising the following steps:

S1: Extraction: add water and halogenated alkanes to the solution containing crude paeoniflorin-6-O'-benzenesulfonate for initial extraction, discard the halogenated alkanes layer; add carboxylate for extraction to obtain carboxylate layer ; Wash the carboxylate layer to obtain the crude product A of paeoniflorin-6-O'-benzenesulfonate;

S2: Separation: the crude product A of paeoniflorin-6-O'-benzenesulfonate is separated by column chromatography to obtain crude product B of paeoniflorin-6-O'-benzenesulfonate with a content of at least 95%;

S3: Recrystallization: recrystallize the crude product B of paeoniflorin-6-O'-benzenesulfonate in a mixed solvent to obtain paeoniflorin-6-O'-benzenesulfonate with a content of at least 98%.

Generally speaking, in the preparation process of paeoniflorin-6-O'-benzenesulfonate, the reaction system of paeoniflorin and benzenesulfonyl chloride is an acetone system, and in the reaction solution after the reaction, according to the present invention, halogenated alkanes are added to it , then the primary extract involves a three-phase system of acetone-water-halogenated alkanes, wherein acetone and water are miscible, acetone and halogenated alkanes are miscible, and water and halogenated alkanes are immiscible, resulting in stratification.

Therefore, the present invention first adopts the three-phase system, so that the impurities in the acetone system enter the haloalkane layer for preliminary impurity removal. The initial extraction can effectively remove

impurities

1, 2, 3, 5 and 8 in the solution containing crude paeoniflorin-6-O'-benzenesulfonate, and obtain paeoniflorin-6- O'-benzenesulfonate crude product A; paeoniflorin-6-O'-benzenesulfonate crude product A can remove impurity 7 after column chromatography and further reduce impurity 4, impurity 6, impurity 9, and impurity 10 , impurity 12, impurity 13 and impurity 14; finally, recrystallization can be used to obviously remove impurity 11 and impurity 15, and the finished product of paeoniflorin-6-O'-benzenesulfonate with a content of at least 98% is obtained.

In some embodiments of the present invention, the content of paeoniflorin-6-O'-benzenesulfonate in the crude paeoniflorin-6-O'-benzenesulfonate described in S1 is at least 70%. For example, at least 75%, preferably at least 80%, more preferably at least 85%; preferably, the crude paeoniflorin-6-O'-benzenesulfonate can be benzenesulfonyl chloride and paeoniflorin and/or benzenesulfonate Acyl chloride is prepared by reacting crude paeoniflorin; more preferably, the crude paeoniflorin-6-O'-benzenesulfonate can be prepared by the prior art or any technique within the range expected by those skilled in the art.

The technical scheme of the present invention provides a ratio range, and the dosage (mass fraction) of acetone is controlled to be 2 to 5 times that of paeoniflorin.

In some preferred embodiments of the present invention, the mass ratio of the solution containing crude paeoniflorin-6-O'-benzenesulfonate to the water in S1 is 1:(2-10).

In some more preferred embodiments of the present invention, the mass ratio of the solution containing crude paeoniflorin-6-O'-benzenesulfonate described in S1 to the halogenated alkanes is 1: (0.1-0.3); further Preferably it is 1:(0.1-0.25). Within this ratio range, the three-phase system can achieve better stratification, achieve obvious impurity removal and further ensure the yield.

In some more preferred embodiments of the present invention, after the initial extraction in S1, the step of secondary extraction is also included before the carboxylate extraction; the secondary extraction is to add haloalkane after the primary extraction Hydrocarbons are extracted; further preferably, the added amount of the halogenated alkanes in the secondary extraction does not exceed 25% of the added amount of the halogenated alkanes in the primary extraction in S1. The secondary extraction is carried out depending on the effect of the primary extraction. When the primary extraction effect is good, the secondary extraction may not be required.

In some more preferred embodiments of the present invention, the halogenated alkanes are selected from chloroform, dichloromethane, carbon tetrachloride, dichloroethane or combinations thereof.

In some more preferred embodiments of the present invention, the carboxylic acid ester in S1 is selected from ethyl acetate, propyl acetate, isopropyl acetate, isobutyl acetate or combinations thereof.

In some more preferred embodiments of the present invention, the mass ratio of the solution containing crude paeoniflorin-6-O'-benzenesulfonate to the carboxylate in S1 is 1: (1-10); Preferably it is 1:(2-8).

In some more preferred embodiments of the present invention, the times of water washing in S1 are 1 to 5 times; more preferably, the times of water washing in S1 are 2 to 3 times.

In some more preferred embodiments of the present invention, the amount of water used for washing in S1 is about 1/2 of the amount of the carboxylate.

In some more preferred embodiments of the present invention, the eluent of the column chromatography in S2 is a mixed solvent of haloalkane-alcohol; further preferably, the haloalkane is selected from chloroform, dichloromethane or a combination thereof; The alcohol is selected from lower alcohols; preferably methanol, ethanol or a combination thereof.

In some more preferred embodiments of the present invention, the volume ratio of the halogenated alkane to the alcohol is (10-15):1; more preferably (10-13):1.

In some more preferred embodiments of the present invention, the mass volume ratio of the crude product A of paeoniflorin-6-O'-benzenesulfonate to the eluent is 1: (45-65) g/mL; More preferably, it is 1:(50-60) g/mL.

In some more preferred embodiments of the present invention, the recrystallization in a mixed solvent described in S3 refers to dissolving the crude product B of paeoniflorin-6-O'-benzenesulfonate in a good solvent, and then adding a poor solvent Perform crystallization.

In some more preferred embodiments of the present invention, the mass volume ratio of the crude product of paeoniflorin-6-O'-benzenesulfonate B to the good solvent is 1: (8-16) g/mL; further Preferably it is 1: (10-13) g/mL.

In some more preferred embodiments of the present invention, the mass volume ratio of the crude paeoniflorin-6-O'-benzenesulfonate B to the poor solvent is 1: (5-8) g/mL.

In some more preferred embodiments of the present invention, the good solvent is selected from any one of isopropyl acetate and isobutyl acetate.

In the present invention, any one of isopropyl acetate and isobutyl acetate is used as a good solvent to recrystallize the crude product B of paeoniflorin-6-O'-benzenesulfonate, which can make paeoniflorin-6-O' - The successful crystallization of benzenesulfonate, and the recrystallization of paeoniflorin-6-O'-benzenesulfonate in a poor solvent or even after crystallization , and its physical state is not conducive to subsequent processing. This is mainly due to the great solubility of paeoniflorin-6-O'-benzenesulfonate in ethyl acetate. When performing poor solvent recrystallization, no matter how the temperature is controlled and the amount of poor solvent is controlled, the precipitation state will always be turbid or viscous paste, and further no matter whether it is taken to stand still, freezing or centrifugation, it is impossible to achieve a good separation of paeoniflorin-6-O'-benzenesulfonate and solvent, which is difficult for subsequent paeoniflorin-6-O'-benzenesulfonate Ester filtration, drying and other processes are extremely unfavorable. On the contrary, by screening a large number of solvent types, dosages, crystallization conditions, etc., the inventor unexpectedly found that only two solvents, isopropyl acetate and isobutyl acetate, can realize the crystallization of paeoniflorin-6-O'-benzenesulfonate. The recrystallization effect, that is, the crystallization can be completed when encountering a poor solvent, and the state is crystal or flocculent, which can be well separated from the solvent system, which is beneficial to the subsequent filtration, drying and other processes.

In some more preferred embodiments of the present invention, the poor solvent is selected from n-hexane, n-heptane, petroleum ether, isopropyl ether, isooctane or combinations thereof.

In some more preferred embodiments of the present invention, the dissolution is heating dissolution, preferably, the temperature of the dissolution is 30°C-70°C.

In some more preferred embodiments of the present invention, the crystallization temperature is 10° C. to 30° C.; more preferably, the crystallization time is not less than 1 h.

In some more preferred embodiments of the present invention, S3 also includes a further purification step after the recrystallization.

In some more preferred embodiments of the present invention, the purification adopts filtration and washing; further preferably, the filtration is selected from any one of vacuum filtration, pressure filtration, and centrifugal filtration; the washing adopts the said poor solvent; more preferably, the number of washings is 2 to 4 times.

The beneficial effects of the present invention are:

1. The present invention adopts a new purification and refining process by improving the reaction conditions, and the purification method of a kind of paeoniflorin-6-O'-benzenesulfonate provided by the invention can effectively remove the preparation of paeoniflorin-6-O'-benzene Benzenesulfonyl chloride and its derivatives, iron, heavy metals and other impurities in the crude sulfonate to obtain paeoniflorin-6-O'-benzenesulfonate with a content higher than 98%.

2. A purification method of paeoniflorin-6-O'-benzenesulfonate provided by the present invention can be used for crude paeoniflorin-6-O' with a paeoniflorin-6-O'-benzenesulfonate content of more than 70%. -Benzenesulfonate is purified to obtain paeoniflorin-6-O'-benzenesulfonate with a content of at least 98%, so it has a wide range of applications and does not have high requirements for the content of raw materials, so it can reduce paeoniflorin-6 -O'-benzenesulfonate production costs.

3. The purification method of paeoniflorin-6-O'-benzenesulfonate provided by the present invention has fewer steps and less time-consuming, and is easy to be industrially applied to the production of paeoniflorin-6-O'-benzenesulfonate.

Description of drawings

The present invention will be further described below in conjunction with accompanying drawing and embodiment, wherein:

Fig. 1 is the HPLC detection result of S1 crude paeoniflorin-6-O'-benzenesulfonate in Test Example 1 of the present invention.

Fig. 2 is the HPLC detection result of each step product of Test Example 1 of the present invention.

Fig. 3 is the influence of different proportions of chloroform on the content and yield of paeoniflorin-6-O'-benzenesulfonate in Test Example 2 of the present invention.

Fig. 4 is the HPLC detection result after refinement of S4 ethyl acetate and isopropyl acetate in Test Example 3 of the present invention.

detailed description

The conception and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments, so as to fully understand the purpose, features and effects of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to The protection scope of the present invention.

Example 1

This embodiment prepared a kind of paeoniflorin-6-O'-benzenesulfonate, the specific process is:

S1: Take 10g of paeoniflorin with a content of 76.3%, add 50mL of acetone, add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains paeoniflorin-6-O'-benzenesulfonate Crude product, the content is 71.5%.

S2: Add 100 mL of purified water and 3 mL of chloroform to the reaction solution for the first extraction, and extract at room temperature for 50 min. After 50 min, the lower layer (chloroform layer) was discarded, and the upper layer (acetone-water) was retained. Add 0.6 mL of chloroform to the reaction solution for a second extraction, extract at room temperature for 30 min, discard the lower layer (chloroform layer), and keep the upper layer (acetone-water). Add 100 mL of ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and keep the organic layer (ethyl acetate). Add 50 mL of purified water to the above ethyl acetate layer and wash with water twice, discard the water layer after standing, and obtain the crude product of paeoniflorin-6-O'-benzenesulfonate 1 with a content of 80.4%, and the extraction step is completed.

S3: The crude product 1 of paeoniflorin-6-O'-benzenesulfonate obtained after extraction is separated by column chromatography, and eluted with chloroform-methanol (10:1) to obtain paeoniflorin-6-O'-benzenesulfonate The crude acid ester 2 has a content of 95.2%.

S4: Heat and dissolve the crude product of paeoniflorin-6-O'-benzenesulfonate 2 with 100mL isobutyl acetate at 65°C, cool down to 25°C after dissolving, add 50mL isopropyl ether, and wait for paeoniflorin-6-O'- After all the benzenesulfonate was precipitated, it was filtered and dried to obtain the final product of paeoniflorin-6-O'-benzenesulfonate with a content of 98.1%.

Example 2

S1: Take 10g of paeoniflorin with a content of 98%, add 50mL of acetone, and simultaneously add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains paeoniflorin-6-O'-benzenesulfonate Crude product, the content is 90.2%.

S2: Add 100 mL of purified water and 3 mL of chloroform to the reaction solution for the first extraction, and extract at room temperature for 50 min. After 50 min, the lower layer (chloroform layer) was discarded, and the upper layer (acetone-water) was retained. Add 0.6 mL of chloroform to the reaction solution for a second extraction, extract at room temperature for 30 min, discard the lower layer (chloroform layer), and keep the upper layer (acetone-water). Add 100 mL of ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and keep the organic layer (ethyl acetate). Add 50 mL of purified water to the above ethyl acetate layer and wash with water twice, discard the water layer after standing, and obtain the crude product of paeoniflorin-6-O'-benzenesulfonate 1 with a content of 94.4%, so far the extraction step is completed.

S3: The crude product 1 of paeoniflorin-6-O'-benzenesulfonate obtained after extraction is separated by column chromatography, and eluted with chloroform-methanol (10:1) to obtain paeoniflorin-6-O'-benzenesulfonate The crude acid ester 2 has a content of 98.3%.

S4: Heat and dissolve the crude product of paeoniflorin-6-O'-benzenesulfonate 2 with 100mL isobutyl acetate at 65°C, cool down to 25°C after dissolving, add 50mL isopropyl ether, and wait for paeoniflorin-6-O'- After all the besylate is precipitated, filter and dry to obtain the final product of paeoniflorin-6-O'-besylate with a content of 99.6%.

Example 3

S1: Take 20g of paeoniflorin with a content of 84.5%, add 120mL of acetone, and at the same time add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains paeoniflorin-6-O'-benzenesulfonate Crude product, the content is 79.2%.

S2: Add 150 mL of purified water and 3 mL of chloroform to the reaction solution for the first extraction, and extract at room temperature for 40 min. After 40 min, the lower layer (chloroform layer) was discarded, and the upper layer (acetone-water) was retained. Add 0.3 mL of chloroform to the reaction solution for a second extraction, extract at room temperature for 20 min, discard the lower layer (chloroform layer), and keep the upper layer (acetone-water). Add 160 mL of ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and keep the organic layer (ethyl acetate). Add 80 mL of purified water to the above ethyl acetate layer and wash with water twice, discard the water layer after standing to obtain the crude product of paeoniflorin-6-O'-benzenesulfonate 1 with a content of 83.9%, and the extraction step is completed.

S3: The crude product 1 of paeoniflorin-6-O'-benzenesulfonate obtained after extraction was separated by column chromatography, and eluted with dichloromethane-methanol (15:1) to obtain paeoniflorin-6-O'- Crude benzenesulfonate 2, the content is 95.7%.

S4: Heat the crude product 2 of paeoniflorin-6-O'-benzenesulfonate with 320mL isobutyl acetate at 55°C to dissolve, cool down to 20°C after dissolving, add 120mL of n-hexane, and wait for paeoniflorin-6-O'-benzene After all the sulfonate was precipitated, it was filtered and dried to obtain the final product of paeoniflorin-6-O'-benzenesulfonate with a content of 98.4%.

Example 4

S1: Take 100g of paeoniflorin with a content of 88.3%, add 500mL of acetone, and at the same time add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains paeoniflorin-6-O'-benzenesulfonate Crude product, the content is 85.6%.

S2: Add 100 mL of purified water and 5 mL of chloroform to the reaction solution for the first extraction, and extract at room temperature for 35 min. After 35 min, the lower layer (chloroform layer) was discarded, and the upper layer (acetone-water) was retained. Add 0.75 mL of chloroform to the reaction solution for the second extraction, extract at room temperature for 10 min, discard the lower layer (chloroform layer), and keep the upper layer (acetone-water). Add 100 mL of ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and keep the organic layer (ethyl acetate). Add 50 mL of purified water to the above ethyl acetate layer and wash twice, discard the water layer after standing to obtain the crude product of paeoniflorin-6-O'-benzenesulfonate 1 with a content of 91.8%, and the extraction step is completed.

S3: The crude product 1 of paeoniflorin-6-O'-benzenesulfonate obtained after extraction is separated by column chromatography, and eluted with chloroform-methanol (13:1) to obtain paeoniflorin-6-O'-benzenesulfonate Crude acid ester 2, the content is 96.0%.

S4: Heat the crude product 2 of paeoniflorin-6-O'-benzenesulfonate with 1200mL isobutyl acetate at 45°C to dissolve, cool down to 10°C after dissolving, add 500mL n-heptane, and wait for paeoniflorin-6-O'- After all the benzenesulfonate was precipitated, it was filtered and dried to obtain the final product of paeoniflorin-6-O'-benzenesulfonate with a content of 98.8%.

Example 5

S1: Take 500g of paeoniflorin with a content of 88.3%, add 2.5L of acetone, and at the same time add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains paeoniflorin-6-O'-benzenesulfonic acid The crude ester has a content of 84.9%.

S2: Add 1.5 L of purified water and 450 mL of chloroform to the reaction solution for the first extraction, and extract at room temperature for 10 min. After 10 min, the lower layer (chloroform layer) was discarded, and the upper layer (acetone-water) was retained. Add 45 mL of chloroform to the reaction solution for the second extraction, extract at room temperature for 15 min, discard the lower layer (chloroform layer), and keep the upper layer (acetone-water). Add 1.5 L of ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and keep the organic layer (ethyl acetate). Add 750 mL of purified water to the above ethyl acetate layer and wash it twice, discard the water layer after standing to obtain the crude product of paeoniflorin-6-O'-benzenesulfonate 1 with a content of 90.6%, and the extraction step is completed.

S3: The crude product 1 of paeoniflorin-6-O'-benzenesulfonate obtained after extraction is separated by column chromatography, and eluted with chloroform-methanol (10:1) to obtain paeoniflorin-6-O'-benzenesulfonate Crude acid ester 2, the content is 95.5%.

S4: Heat and dissolve the crude product of paeoniflorin-6-O'-benzenesulfonate 2 with 5L of isopropyl acetate at 35°C, cool down to 15°C after dissolving, add 3L of isopropyl ether, and wait for paeoniflorin-6-O'- After all the benzenesulfonate was precipitated, it was filtered and dried to obtain the final product of paeoniflorin-6-O'-benzenesulfonate with a content of 98.6%.

Example 6

S1: Take 1 kg of paeoniflorin with a content of 92.5%, add 5 L of acetone, and simultaneously add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains paeoniflorin-6-O'-benzenesulfonate Crude product, the content is 88.1%.

S2: Add 5L of purified water and 750mL of chloroform to the reaction solution for the first extraction, and extract at room temperature for 15 minutes. After 15 min, the lower layer (chloroform layer) was discarded and the upper layer (acetone-water) was retained. Add 37.5 mL of chloroform to the reaction solution for the second extraction, extract at room temperature for 20 min, discard the lower layer (chloroform layer), and keep the upper layer (acetone-water). Add 5 L of ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and keep the organic layer (ethyl acetate). Add 2.5 L of purified water to the above ethyl acetate layer and wash with water twice, discard the water layer after standing to obtain the crude product of paeoniflorin-6-O'-benzenesulfonate 1 with a content of 93.1%, and the extraction step is completed.

S3: The crude product 1 of paeoniflorin-6-O'-benzenesulfonate obtained after extraction is separated by column chromatography, and eluted with chloroform-methanol (14:1) to obtain paeoniflorin-6-O'-benzenesulfonate The crude acid ester 2 has a content of 97.5%.

S4: Heat and dissolve the crude product 2 of paeoniflorin-6-O'-benzenesulfonate in 12L of isopropyl acetate at 40°C, cool down to 25°C after dissolving, add 7L of isooctane, and wait for paeoniflorin-6-O'- After all the besylate is precipitated, filter and dry to obtain the final product of paeoniflorin-6-O'-besylate with a content of 99.3%.

Example 7

S1: Take 10kg of paeoniflorin with a content of 92.5%, add 50L of acetone, and at the same time add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains paeoniflorin-6-O'-benzenesulfonate Crude product, the content is 87.4%.

S2: Add 100L of purified water and 3L of chloroform to the reaction solution for the first extraction, and extract at room temperature for 60 minutes. After 60 min, the lower layer (chloroform layer) was discarded, and the upper layer (acetone-water) was retained. Add 0.75 L of chloroform to the reaction solution for the second extraction, extract at room temperature for 60 min, discard the lower layer (chloroform layer), and keep the upper layer (acetone-water). Add 100 L of ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and keep the organic layer (ethyl acetate). Add 50 L of purified water to the above ethyl acetate layer and wash with water twice, discard the water layer after standing to obtain the crude product of paeoniflorin-6-O'-benzenesulfonate 1 with a content of 92.5%, and the extraction step is completed.

S3: The crude product 1 of paeoniflorin-6-O'-benzenesulfonate obtained after extraction was separated by column chromatography, and eluted with dichloromethane-methanol (12:1) to obtain paeoniflorin-6-O'- Crude benzenesulfonate 2, the content is 96.9%.

S4: Heat and dissolve the crude product of paeoniflorin-6-O'-benzenesulfonate 2 with 80L isopropyl acetate at 45°C, cool down to 30°C after dissolving, add 80L of n-hexane, and wait for paeoniflorin-6-O'-benzene After all the sulfonate is precipitated, filter and dry to obtain the final product of paeoniflorin-6-O'-benzenesulfonate with a content of 99.0%.

Example 8

S1: Take 100kg of paeoniflorin with a content of 90%, add 500L of acetone, and at the same time add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains paeoniflorin-6-O'-benzenesulfonate Crude product, the content is 85.4%.

S2: Add 1000L of purified water and 30L of chloroform to the reaction solution for the first extraction, and extract at room temperature for 60 minutes. After 60 min, the lower layer (chloroform layer) was discarded, and the upper layer (acetone-water) was retained. Add 7.5 L of chloroform to the reaction solution for the second extraction, extract at room temperature for 60 min, discard the lower layer (chloroform layer), and keep the upper layer (acetone-water). Add 1000L ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and keep the organic layer (ethyl acetate). Add 500L of purified water to the above ethyl acetate layer and wash with water twice, discard the water layer after standing to obtain the crude product of paeoniflorin-6-O'-benzenesulfonate 1 with a content of 91.4%, and the extraction step is completed.

S3: The crude product 1 of paeoniflorin-6-O'-benzenesulfonate obtained after extraction was separated by column chromatography, and eluted with dichloromethane-methanol (12:1) to obtain paeoniflorin-6-O'- Crude benzenesulfonate 2, the content is 96.5%.

S4: Heat and dissolve the crude product of paeoniflorin-6-O'-benzenesulfonate 2 in 800L isopropyl acetate at 55°C, cool down to 30°C after dissolving, add 500L isooctane, and wait for paeoniflorin-6-O'- After all the benzenesulfonate was precipitated, it was filtered and dried to obtain the final product of paeoniflorin-6-O'-benzenesulfonate with a content of 98.7%.

Test example 1

In this test example, the impurity removal effect during the refining process of paeoniflorin-6-O'-benzenesulfonate was monitored, and samples were taken after the reaction, primary extraction, secondary extraction, column chromatography separation, and recrystallization. Carry out HPLC monitoring, focus on monitoring the changes of impurities in the material, the results are shown in Figure 1 and Figure 2. The specific process is:

S1: Take paeoniflorin (containing 1g paeoniflorin) with a content of 90%, add 4mL of acetone, and add benzenesulfonyl chloride, triethylamine, etc. to react at the same time, and the obtained reaction solution contains paeoniflorin-6-O'-benzenesulfonate The crude acid ester was sampled for HPLC monitoring.

S2: Add 4.5 mL of purified water and 0.2 mL of chloroform to the reaction solution for the first extraction, and extract at room temperature for 30 min. After 30 min, the lower layer (chloroform layer) was discarded, the upper layer (acetone-water) was retained, and samples were taken for HPLC monitoring. Then add 0.04mL chloroform to the reaction solution for the second extraction, extract at room temperature for 30min, discard the lower layer (chloroform layer), continue to keep the upper layer (acetone-water), and sample for HPLC monitoring. Add 5 mL of ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and keep the organic layer (ethyl acetate). 2.5 mL of purified water was added to the ethyl acetate layer to wash twice, and the water layer was discarded after standing to obtain the crude product of paeoniflorin-6-O'-benzenesulfonate 1, and the extraction step was completed.

S3: The crude paeoniflorin-6-O'-benzenesulfonate 1 obtained after extraction was separated by column chromatography to obtain the crude paeoniflorin-6-O'-benzenesulfonate 2, which was sampled for HPLC monitoring.

S4: Heat and dissolve the crude product of paeoniflorin-6-O'-benzenesulfonate 2 in 15 mL of isopropyl acetate at 50 °C, cool down to 30 °C after dissolving, add 7.5 mL of n-hexane, and wait for paeoniflorin-6-O'- After all the benzenesulfonate was precipitated, it was filtered and dried to obtain the final product of paeoniflorin-6-O'-benzenesulfonate, which was sampled for HPLC monitoring.

Figure 1 and Figure 2 show that after the paeoniflorin extract reacts with benzenesulfonyl chloride, 15 impurities are generated in total, of which, except impurity 7 is a known impurity (paeonifloride-6-O'-benzenesulfonate), The remaining 14 kinds of impurities are unknown impurities after the reaction. From the research results, the extraction process of the technical solution of the present invention has a good removal effect on the

impurities

1, 2, 3, 5 and 8 after the paeoniflorin-6-O'-benzenesulfonate reaction . After extraction, the types of impurities decreased from 15 to 11, and impurity 1 decreased from 3.69% to 0.23%. Overall, the extraction method can obviously remove the impurity species before the main peak, but the removal effect of the impurity species after the main peak is poor (except for impurity 8). It can be seen that the refining effect brought by the extraction method is mainly concentrated before the main peak .

After extraction, the content of paeoniflorin-6-O'-benzenesulfonate can reach more than 91%, but the impurities that are completely removed are only impurity 2, impurity 3, impurity 5, and impurity 8. Impurity 1, impurity 4, impurity 9, impurity 10, impurity 12 and impurity 13 can be further removed by further column chromatography separation. The impurity 10 was reduced from 0.44% after extraction to 0.13% after column chromatography. Column chromatography separation is mainly to remove impurity 7, namely paeonifloride-6-O'-benzenesulfonate. The results showed that the impurity 7 was reduced from 1.06% to 0.48% by column chromatography. Other impurities were all reduced to below the detection limit due to their small content. After column chromatography, the content of paeoniflorin-6-O'-benzenesulfonate reached more than 95%, and the types of impurities were further reduced from the original 15 to 6. The research shows that the column chromatography process is mainly to reduce the impurity 7, that is, paeonifloride-6-O'-benzenesulfonate, and can further reduce the

impurities

4, 6, 9, 10, 12, and 13 , The content of impurity 14.

It can be clearly seen from the above results that neither extraction nor column chromatography can effectively reduce impurity 11 and impurity 15. Therefore, in order to obtain paeoniflorin-6-O'-benzenesulfonate with a content greater than 98%, it is necessary to add a recrystallization step to remove impurity 11 and impurity 15. After recrystallization, the content of impurity 11 and impurity 15 decreased significantly, wherein impurity 15 could be completely removed, and the content of impurity 11 decreased significantly from the original 2.56% to 0.65%. Paeoniflorin-6-O'-benzenesulfonate The content reaches more than 99%.

In summary, the purification method of paeoniflorin-6-O'-benzenesulfonate of the present invention can finally obtain the finished product of paeoniflorin-6-O'-benzenesulfonate with a content of more than 98%. Among them, the extraction technology has obvious removal effect on

impurities

1, 2, 3, 5, and 8, and column chromatography can further reduce the content of

impurities

4, 6, 9, 10, 12, 13, and 14. The removal effect of ester glycoside-6-O'-benzenesulfonate was the most obvious, while

impurities

11, 14 and 15 were further reduced by means of recrystallization.

Test example 2

The technical scheme of the present invention provides a purification method of paeoniflorin-6-O'-benzenesulfonate, which involves a solvent extraction step. It can be known from the above test example 1 that the extraction process can effectively remove the impurities before the main peak and the impurity 8. Considering the extraction process, the whole system is a ternary system involving reaction solvent, extraction solvent and purified water. Because acetone and water are soluble in each other, acetone and halogenated alkanes are soluble in each other, and water and halogenated alkanes are not. Therefore, adjusting the ratio of acetone-water-halogenated alkanes can effectively improve the effect of initial extraction, so that it can be well separated and ensure obvious impurity removal and higher yield. Therefore, this test example investigates the proportion of halogenated alkanes in acetone-water-halogenated alkanes, and uses the content and yield as the investigation index to screen the optimal amount of halogenated alkanes.

According to the test scheme of Test Example 1, taking chloroform as an example, by the single factor investigation method, carry out the screening of the suitable dosage ratio of halogenated alkanes in the extraction process. Based on the total volume of chloroform and purified water, the amount of chloroform added was set at different ratios of 5%, 10%, 15%, 20%, 25%, and 30%. Considering that the recrystallization process is mainly aimed at impurity 11 and impurity 15, and has little impact on the yield, so the test procedure ends after column chromatography, and other operations are the same as effect example 1, and the yield and content of samples obtained in different groups are measured respectively. The result is shown in Figure 3.

According to the results in Fig. 3, it can be clearly seen that the proportion of chloroform presents a negative correlation trend for the content and yield of paeoniflorin-6-O'-benzenesulfonate (samples after column chromatography). With the increase of the amount of chloroform, the extraction effect was obviously improved, but the yield dropped sharply at the same time. The main reason for adopting chloroform for extraction is that the impurities before the main peak can be dissolved in chloroform, but at the same time, paeoniflorin-6-O'-benzenesulfonate can also be dissolved in chloroform, so as the amount of chloroform increases, more and more Many impurities were dissolved in the chloroform layer, but at the same time a large amount of paeoniflorin-6-O'-benzenesulfonate could be dissolved in chloroform. This has just caused, along with the increase of chloroform consumption, sample content is obviously improved, but yield also drops sharply. Therefore, in order to balance the relationship between yield and content, it is necessary to adopt a suitable chloroform ratio.

It can be clearly seen from Figure 3 that when the proportion of chloroform exceeds 25%, the degree of increasing the content of paeoniflorin-6-O'-benzenesulfonate is significantly reduced, for example, when the amount of chloroform is 25%, paeoniflorin-6-O'- The content of benzenesulfonate is 96.13%, and when the proportion of chloroform is continuously increased to 30%, its content is 96.86%, and the content is only increased by 0.73%, but the yield is lost by nearly 20%. Therefore, from the perspective of content and yield, the ratio of chloroform in the acetone-water-chloroform three-phase system is preferably controlled at 10% to 25%, and 30% chloroform can also meet the content requirements, but its yield is relatively low. lower.

Test example 3

The technical scheme of the present invention provides a method for purifying paeoniflorin-6-O'-benzenesulfonate, which involves a solvent refining step. It can be known from the above test example 1 that the extraction process can effectively remove

impurities

11, 14 and 15. Use any one of isopropyl acetate and isobutyl acetate as a good solvent to recrystallize the crude product B of paeoniflorin-6-O'-benzenesulfonate, which can make paeoniflorin-6-O'-benzenesulfonic acid The ester was successfully crystallized, but the recrystallization using conventional solvents such as ethyl acetate will cause paeoniflorin-6-O'-benzenesulfonate to fail to crystallize in a poor solvent or its physical state after precipitation is turbid or Viscous paste, and no matter whether it is taken to stand still, freeze or centrifuge, it is impossible to achieve a good separation of paeoniflorin-6-O'-benzenesulfonate and solvent, and it is difficult to separate paeoniflorin-6-O'-benzenesulfonate after refining. The content of besylate is raised to more than 98% (as shown in Figure 4).

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those of ordinary skill in the art without departing from the gist of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other if there is no conflict.

Claims

A method for purifying paeoniflorin-6-O'-benzenesulfonate, characterized in that it comprises the following steps:

S1: Extraction: add water and halogenated alkanes to the solution containing crude paeoniflorin-6-O'-benzenesulfonate for initial extraction, discard the halogenated alkanes layer; add carboxylate for extraction to obtain carboxylate layer ; Wash the carboxylate layer to obtain the crude product A of paeoniflorin-6-O'-benzenesulfonate;

S2: Separation: the crude product A of paeoniflorin-6-O'-benzenesulfonate is separated by column chromatography to obtain crude product B of paeoniflorin-6-O'-benzenesulfonate with a content of at least 95%;

S3: Recrystallization: recrystallize the crude product B of paeoniflorin-6-O'-benzenesulfonate in a mixed solvent to obtain paeoniflorin-6-O'-benzenesulfonate with a content of at least 98%.
The purification method of paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: in S3, said recrystallization in a mixed solvent refers to said paeoniflorin-6-O' - After the crude benzenesulfonate ester B is dissolved in a good solvent, a poor solvent is added for crystallization.
The purification method of paeoniflorin-6-O'-benzenesulfonate according to claim 2, characterized in that: the good solvent is selected from isopropyl acetate, isobutyl acetate or a combination thereof.
The purification method of paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: in the crude paeoniflorin-6-O'-benzenesulfonate described in S1, paeoniflorin-6- The content of O'-benzenesulfonate is at least 70%; preferably, the solution containing crude paeoniflorin-6-O'-benzenesulfonate refers to an acetone solution.
The purification method of paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: the solution containing crude paeoniflorin-6-O'-benzenesulfonate described in S1 and the The mass ratio of water is 1: (2-10).
The purification method of paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: the solution containing crude paeoniflorin-6-O'-benzenesulfonate described in S1 and the The mass ratio of halogenated alkanes is 1: (0.1-0.3).
The purification method of paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: after the initial extraction in S1, before the extraction of carboxylate, the step of secondary extraction is also included; The secondary extraction is performed by adding halogenated alkanes after the initial extraction.
The purification method of paeoniflorin-6-O'-benzenesulfonate according to claim 1 or 5, characterized in that: the halogenated alkane is selected from chloroform, dichloromethane, carbon tetrachloride, dichloroethane or a combination thereof.
The purification method of paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: the solution containing crude paeoniflorin-6-O'-benzenesulfonate described in S1 and the The mass ratio of the carboxylate is 1:(1-10); the carboxylate is preferably ethyl acetate, propyl acetate, or butyl acetate.
The purification method of paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: the eluent of the column chromatography in S2 is a halogenated alkane-alcohol mixed solvent.
The purification method of paeoniflorin-6-O'-benzenesulfonate according to claim 10, characterized in that: the volume ratio of the halogenated alkane to the alcohol is (8-16):1.
The purification method of paeoniflorin-6-O'-benzenesulfonate according to claim 2, characterized in that: the poor solvent is selected from normal hexane, normal heptane, petroleum ether, isopropyl ether, isooctane or a combination thereof.
The purification method of paeoniflorin-6-O'-benzenesulfonate according to claim 2, characterized in that: the mass of the crude product B of paeoniflorin-6-O'-benzenesulfonate and the good solvent The volume ratio is 1:(8~16)g/mL.
The purification method of paeoniflorin-6-O'-benzenesulfonate according to claim 2, characterized in that: the mass of the crude product B of paeoniflorin-6-O'-benzenesulfonate and the poor solvent The volume ratio is 1: (5-8) g/mL.