WO2023236453A1

WO2023236453A1 - Method for preparing albiflorin-6'-o-benzenesulfonate

Info

Publication number: WO2023236453A1
Application number: PCT/CN2022/133498
Authority: WO
Inventors: 白柏; 许文东; 刘菊妍; 唐顺之; 袁诚; 韩亚明; 彭万才; 梁北梅; 魏劭恒; 傅玉萍; 李佳俐
Original assignee: 广州白云山汉方现代药业有限公司
Priority date: 2022-06-09
Filing date: 2022-11-22
Publication date: 2023-12-14
Also published as: CN115677804A

Abstract

The present invention belongs to the field of compounds, and discloses a method for preparing albiflorin-6'-O-benzenesulfonate. The method comprises the following steps: taking a medicinal material that is rich in albiflorin as a starting material, subjecting same to extraction, adsorption, separation and purification to obtain albiflorin with a relatively high purity; and reacting an organic metal catalyst, an acid-binding agent and chemically active benzenesulfonyl chloride with albiflorin, carrying out column chromatography separation and refining on the product to obtain more than 98% of albiflorin-6'-O-benzenesulfonate, dissolving same in a mixed solvent, and carrying out programmed drying to finally obtain a finished product of albiflorin-6'-O-benzenesulfonate. The method has a high reaction yield, a simple operation, and mild conditions, and is very suitable for industrial production of albiflorin-6'-O-benzenesulfonate.

Description

A kind of preparation method of paeoniflorin-6’-O-benzenesulfonate

Technical field

The invention belongs to the field of compounds, and specifically relates to a preparation method of paeoniflorin-6'-O-benzenesulfonate.

Background technique

White peony root is the root of Paeonia lactiflora, which has antipyretic and analgesic effects. Modern pharmacology shows that white peony extract also has various pharmacological activities such as anti-inflammatory and immunomodulatory, detoxifying and liver-protecting, anti-mutagenic and anti-tumor. Total glycosides of white peony are also the first to be used in the clinical treatment of rheumatoid joints. It is an anti-inflammatory immunomodulatory drug for inflammation, and has the characteristics of good curative effect, few adverse reactions, and good tolerance. As one of the main active ingredients of white peony root, Albiflorin also has a variety of pharmacological effects, such as anti-epileptic, analgesic, detoxification, anti-vertigo, etc. It can also be used to treat rheumatoid arthritis and bacterial infections. Dysentery, enteritis, viral hepatitis, senile diseases, etc.

Paeoniflorin is the second most abundant active ingredient in the traditional Chinese medicine white peony root [1]. In recent years, with in-depth research on its pharmacodynamics, it has been found that paeoniflorin glycosides have unique pharmacodynamic characteristics that are different from paeoniflorin. It has better pharmacological effects than paeoniflorin in terms of blood enrichment, antidepressant, and potential treatment of diabetes. characteristics, and has anti-convulsant, anti-inflammatory and liver-protecting effects [2]. Furthermore, in the study of the pharmacokinetics of paeoniflorin glycosides, it was found that after oral administration of paeoniflorin glycosides to ICR mice, except for the stomach, intestine, and kidney, the contents of other tissues were lower and the clearance rate was rapid [3]. This is It may be related to the poor fat solubility of paeoniflorin glycosides. Therefore, it is necessary to carry out derivatization of paeoniflorin glycosides to improve the fat solubility of paeoniflorin glycosides, thereby improving the absorption and bioavailability of paeoniflorin glycosides and better exerting its medicinal effects.

Patent CN102603827 studies the preparation method of the similar compound paeoniflorin-6'-O-benzenesulfonate, using 98% paeoniflorin as the raw material, using pyridine as the acid compress and reaction solvent, DMAP as the catalyst, and slowly adding peony root. When the molar ratio of glycosides to benzene sulfonyl chloride dissolved in methylene chloride is 20 hours, after post-treatment, the yield is about 40%. The compound paeoniflorin-6'-O-benzenesulfonate described in this patent can also be synthesized by this method, but there are the following shortcomings: 1. The purity of paeoniflorin required for the reaction raw materials is not less than 98%. Sources are limited, and there are currently no kilogram-level raw materials with a purity of more than 98% on the market; 2. The reaction yield is low, the catalyst, reaction solvent and subsequent purification solvent required for the reaction are large, and the reaction process is not economical and environmentally friendly; 3. The reaction raw materials need to be added step by step, and the raw materials need to be added slowly during the reaction process. The reaction time for large-scale preparation is long and the process is complicated, which is not conducive to industrial production.

On the other hand, because paeoniflorin-6'-O-benzenesulfonate has the characteristics of strong lipophilicity, difficulty in crystallization, strong hygroscopicity, and poor thermal stability, it significantly increases the difficulty of the drying process. After adopting the process of directly drying the precipitates, the solvent encapsulation phenomenon exists in the finished product, causing the solvent residue to seriously exceed the standard. If the sample is dissolved and dried directly under vacuum, paeoniflorin-6'-O-benzenesulfonate is prone to alcohol degradation, resulting in a significant increase in related substances, which undoubtedly brings challenges to the research on drying processes. Using the principle of sublimation, the material is heated and dried under low temperature and vacuum conditions, which is suitable for the preparation of heat-sensitive materials. Materials that are soluble and stable in aqueous solution can use water as the sole solvent for this drying procedure, but paeoniflorin-6'-O-benzenesulfonate is not suitable because it is slightly soluble in water. Tert-butanol, which has high melting point, high vapor pressure, non-toxicity and is miscible with water, is an excellent choice as a drying solvent for drugs that are difficult to dissolve in water and are heat-labile. Tert-butanol-water co-solvent has been used in a variety of injectable drugs. The production process can improve drying efficiency and shorten drying time, but paeoniflorin-6'-O-benzenesulfonate is unstable in alcohol-containing solvents and prone to degradation, resulting in a greatly reduced quality of the finished product that does not meet the requirements.

Therefore, in order to solve the above problems, it is urgent to develop a process route suitable for the industrial preparation of paeoniflorin-6'-O-benzenesulfonate.

Contents of the invention

The present invention provides for the first time a preparation route for paeoniflorin-6'-O-benzenesulfonate. Using medicinal materials rich in paeoniflorin such as white peony root as raw materials, 80% is obtained through extraction, adsorption and silica gel column chromatography. % or more of paeoniflorin glycosides, through esterification reaction, obtain the crude paeoniflorin glycoside-6'-O-benzenesulfonate, and then through chromatographic column separation, purification and drying, finally obtain the medicinal paeoniflorin glycosides -6'-O-benzenesulfonate.

In order to solve the above technical problems, the present invention adopts the following technical solutions:

A preparation method of paeoniflorin-6’-O-benzenesulfonate, comprising the following steps:

S1: Extract paeoniflorin glycosides from medicinal materials;

S2: Add an organic metal catalyst, an acid binding agent, and benzenesulfonyl chloride to the paeoniflorin glycoside in proportion to carry out the reaction. After the reaction is terminated, the reaction product is concentrated to obtain a crude paeoniflorin glycoside-6'-O-benzenesulfonate ester;

S3: Separate the crude paeoniflorin-6'-O-benzenesulfonate product to obtain paeonilide-6'-O-benzenesulfonate fine product A;

S4: After dissolving the paeoniflorin-6'-O-benzenesulfonic acid ester boutique A in a good solvent, add a poor solvent for crystallization, and obtain paeoniflorin-6'-O-benzenesulfonic acid after crystallization. Ester Boutique B;

S5: Dissolve the paeoniflorin-6'-O-benzenesulfonate boutique B in a mixed solvent, the mixed solvent includes dimethyl carbonate, C3~4 ketones and water, and perform programmed drying. Finally, the finished product of paeoniflorin-6'-O-benzenesulfonate is obtained.

According to the method of the present invention, an organic metal catalyst, an acid binding agent and a chemically active benzenesulfonyl chloride are used to react with paeoniflorin glycoside in S2, so that the reaction can produce paeoniflorin glycoside-6'-O-benzenesulfonic acid. The ester reaches more than 80%, and then is separated and purified by column chromatography and then refined to obtain more than 98% of paeoniflorin-6'-O-benzenesulfonate ester.

After the refining in step S4, the purity of the obtained paeoniflorin-6'-O-benzenesulfonate fine product B has reached more than 98% after testing. However, since in the purity test, the liquid phase solvent does not produce peaks or is subtracted from the background, premium product B actually contains more solvents used in the refining process. According to the provisions of the Pharmacopoeia, the residues of various solvents used in the present invention in the finished drug should basically be controlled below 0.5%, so further drying of S5 is required.

In step S5, the present invention proposes a ternary solvent system of dimethyl carbonate, C3-4 ketones, and water with a proportion within a certain range. The freezing point is -10-0°C, which meets the solvent requirements of the programmed drying process and is expected to Unexpectedly, while successfully solving the drying problem of fat-soluble paeoniflorin-6'-O-benzenesulfonate, the related substances in the finished product no longer increase compared with before drying, and the solvent residues (including: the good quality used for refining The solvent isopropyl acetate or isobutyl acetate, the drying solvent dimethyl carbonate, acetone) can all be lower than the specified value.

In some embodiments of the present invention, the mixed solvent consists of dimethyl carbonate, acetone and water, or consists of dimethyl carbonate, butanone and water. Under this combination, the mixed solvent has better solubility for paeoniflorin-6'-O-benzenesulfonate, which can better reduce solvent residues and reduce the increase in related substance content after the drying process.

In some embodiments of the present invention, after programmed drying, the total solvent residue of paeoniflorin-6'-O-benzenesulfonate is no more than 0.6%. Preferably, after programmed drying, the carbonate residue of paeoniflorin-6'-O-benzenesulfonate is not higher than 0.5%, and the residue of C3~4 ketones is not higher than 0.2%.

In some embodiments of the present invention, after programmed drying, the content of related substances in paeoniflorin-6'-O-benzenesulfonate increases by no more than 0.2%.

Preferably, it includes the following steps:

S1: Includes the following steps:

S1-1: The medicinal materials are extracted with alcohol, the extract is filtered and concentrated to obtain the medicinal material extract, and the crude paeoniflorin glycoside is obtained through adsorption and elution;

S1-2: The crude paeoniflorin glycoside is separated by column chromatography to obtain the paeoniflorin glycoside, and the purity of the paeoniflorin glycoside is above 80%;

S2: Add the organometallic catalyst, the acid binding agent, and the benzenesulfonyl chloride to the paeoniflorin glycoside in proportion, carry out the reaction in the reaction solvent, and then add the organic phase and the aqueous phase to terminate the reaction and perform extraction. , after concentration, the crude paeoniflorin-6'-O-benzenesulfonate ester is obtained;

S3: Separate the crude paeoniflorin-6'-O-benzenesulfonate ester through column chromatography, collect the eluent, and obtain the paeonilide-6'-O-benzenesulfonate fine product A , the purity of the paeoniflorin-6'-O-benzenesulfonate fine product A is above 95%;

S4: After dissolving the paeoniflorin-6'-O-benzenesulfonate fine product A with the good solvent, adding the poor solvent for crystallization, and obtaining paeonilide-6'-O after crystallization. -Exquisite benzenesulfonate B, the purity of the paeoniflorin-6'-O-benzenesulfonate elite B is above 98%;

S5: Dissolve the paeoniflorin-6'-O-benzenesulfonate boutique B in the mixed solvent, which includes dimethyl carbonate, C3~4 ketones and water in a volume ratio of 4 ~11:0.2~0.6:1 is mixed, and programmed drying is performed to finally obtain the finished product of paeoniflorin-6'-O-benzenesulfonate.

In steps S1-2 and S3, those skilled in the art know that when column chromatography is used for separation, the starting point for collecting the eluent is when the main component appears, and the end point is when impurities appear or the spots of the main component become smaller.

Preferably, in step S1-1, the medicinal materials include one or more of white peony root, red peony root, paeoniae bark, dog head, peony root, and purple peony root, using 80% to 100% ethanol or 80% to 100% ethanol. Methanol extraction, the extraction method includes one of cold soaking, thermal reflux or microwave-assisted extraction; in step S1-2, the column chromatography separation is silica gel column chromatography separation, and the eluent is an alkane derivative-lower alcohol mixed solvent ; In step S2, the organometallic catalyst includes one or more of C1 to C4 alkyl tin halides, aryl tin halides, C1 to C4 alkyl tin oxides, and C1 to C4 alkyl tin sulfides, The acid binding agent includes one or more of triethylamine, N, N'-diisopropylethylamine, pyridine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate, and the reaction solvent includes One or more of acetonitrile, tetrahydrofuran, acetone, pyridine, methyl ethyl ketone, N,N'-dimethylformamide, 1,4-dioxane, and dimethyl sulfoxide, the organic phase is ethyl acetate; in step S3, the column chromatography separation is silica gel column chromatography separation, and the eluent is a halogenated hydrocarbon-lower alcohol mixed solvent; in step S4, the good solvent includes isopropyl acetate, isopropyl acetate One or more of butyl ester, the poor solvent includes one or more of n-hexane, petroleum ether, isopropyl ether, isooctane; in step S5, the mixed solvent includes dimethyl carbonate, C3 ~4 Ketones and water are mixed in a volume ratio of 5~8:0.3~0.5:1.

In step S1-2, compared with the haloalkane-alcohol system, when the alkane derivative-alcohol system is used in the present invention to separate the crude paeoniflorin glycosides by column chromatography, its elution capacity is stronger, and the separation effect is achieved at the same time , the amount of eluent is relatively small, and it is less toxic than chloroform, making the production safer and more environmentally friendly.

In step S2, chemically active benzenesulfonyl chloride is used as the substrate. In order to improve the safety of the reaction, it needs to be used under anhydrous conditions. Pyridine can be used as both a reaction solvent for S2 and an acid-binding agent. However, pyridine is reproductively toxic and is not suitable for large-scale industrial use. From the perspective of low toxicity and low cost, in industrial production, the reaction solvent can be organic solvents such as acetone and acetonitrile, and the acid binding agent can be organic acid binding agents such as triethylamine, or inorganic acid binding agents such as potassium carbonate.

In some embodiments of the present invention, S2 also includes extracting the product with ethyl acetate and water after the reaction is terminated, and then concentrating to obtain the crude paeoniflorin-6'-O-benzenesulfonate ester. . The reaction temperature was room temperature, and the reaction time was 2 to 5 hours. It is best to use artificial control to terminate the reaction process. If the reaction is not terminated and the reaction time is too long, more impurities will be produced by side reactions. Add water and ethyl acetate and stir to terminate the reaction and perform extraction at the same time. After stirring, let it stand and separate the layers to take the organic layer. After the reaction in S2, there will generally be by-products, organic salts, acid binding agents and incompletely reacted substrates. Using ethyl acetate and water to extract the extract can effectively remove some of the by-products and other impurities, thereby A crude paeoniflorin-6'-O-benzenesulfonate product with higher purity is obtained.

Experiments have found that if you do not add a poor solvent, directly add a good solvent to dissolve Paeoniflorin-6'-O-benzenesulfonate Premium A and then cool it down to crystallize, you will find that you cannot obtain solid crystals and can only obtain a viscous syrupy state. Substance, solid-liquid separation cannot be separated by filtration, which may be related to the special properties of paeoniflorin-6'-O-benzenesulfonate. Therefore, the present invention creatively uses the method of dissolving paeoniflorin-6'-O-benzenesulfonate fine product A in a good solvent and then adding a poor solvent for crystallization in step S4, which can improve the purity of the product and reduce the Impurities.

Preferably, in step S1-1, the medicinal material includes one or more of white peony root and red peony root, and is extracted using 95% to 100% ethanol or 95% to 100% methanol. The extraction method includes thermal reflux; Step S1 -2, the mass ratio of the silica gel used for the silica gel column chromatography separation to the crude paeoniflorin glycoside is 10 to 20:1, and the mass ratio of the alkane derivative-lower alcohol mixed solvent to the crude paeoniflorin glycoside The volume to mass ratio is 40 to 65 ml: 1 g; in step S2, the dosage of the benzenesulfonyl chloride is 20% to 60% of the mass of the paeoniflorin glycoside, and the dosage of the organic metal catalyst is the paeoniflorin glycoside. 0.05% to 20% of the mass, the molar ratio of the acid binding agent to the paeoniflorin glycoside is 1 to 2:1, and the volume to mass ratio of the reaction solvent to the paeoniflorin glycoside is 2 to 10 ml: 1g, the volume and mass ratio of the organic phase and the paeoniflorin glycoside is 2.5-7.5ml: 1g, the volume and mass ratio of the aqueous phase and the paeoniflorin glycoside is 5-15ml: 1g; in step S3 , the mass ratio of the silica gel used for the silica gel column chromatography separation to the crude paeoniflorin-6'-O-benzenesulfonate is 6 to 12:1, and the halogenated hydrocarbon-lower alcohol mixed solvent and the crude paeoniflorin-6'-O-benzenesulfonate are The volume to mass ratio of the crude paeoniflorin-6'-O-benzenesulfonate is 55 to 80 ml: 1 g; in step S4, the good solvent and the paeonilide-6'-O-benzenesulfonate are The volume-to-mass ratio of the fine acid ester A is 10 to 20 mL: 1 g, and the volume-to-mass ratio of the poor solvent to the fine paeoniflorin-6'-O-benzenesulfonate ester A is 8 to 12 mL: 1g; steps In S5, the volume to mass ratio of the mixed solvent and the paeoniflorin-6'-O-benzenesulfonate fine product B is 1 to 20 mL: 1 g.

Preferably, in step S1-1, the mass ratio of ethanol or methanol to the medicinal material is 5 to 20:1; in step S1-2, the mass of the silica gel used for silica gel column chromatography separation and the crude paeoniflorin glycosides The ratio is 10-15:1, and the volume-to-mass ratio of the alkane derivative-lower alcohol mixed solvent and the crude paeoniflorin glycoside is 50-60ml:1g; in step S2, the amount of benzene sulfonyl chloride is 25% to 30% of the mass of the paeoniflorin glycoside, the dosage of the organometallic catalyst is 0.5% to 5.0% of the mass of the paeoniflorin glycoside, and the molar ratio of the acid binding agent to the paeoniflorin glycoside It is 1.2～1.6:1; in step S5, the volume mass ratio of the mixed solvent and the paeoniflorin-6'-O-benzenesulfonate fine product B is 1～15mL:1g.

In step S1-2, when the amount of filler is larger, the separation effect is better. When the amount is 10 to 15 times that of the crude paeoniflorin glycoside, the amount of eluent is relatively small while ensuring a better separation effect. .

Preferably, in step S1-1, the mass ratio of ethanol or methanol to the medicinal material is 5 to 10:1; in step S1-2, the mesh number of the silica gel used for silica gel column chromatography separation is 60 to 400 mesh, and the pore size is 60 to 400 mesh. for

The volume ratio of the alkane derivative to the lower alcohol in the alkane derivative-lower alcohol mixed solvent is 1 to 10:1; in step S2, the amount of the organometallic catalyst is 0.6% to 0.6% of the mass of the paeoniflorin glycosides. 3.0%; in step S3, the mesh number of the silica gel used for the silica gel column chromatography separation is 60 to 400 mesh, and the pore diameter is

The volume ratio of the halogenated hydrocarbon to the lower alcohol in the halogenated hydrocarbon-lower alcohol mixed solvent is 6 to 12:1; in step S4, the paeoniflorin-6'-O-benzenesulfonate fine product A After dissolving with the good solvent at 50-60°C, the temperature is lowered to 10-20°C and the poor solvent is added for crystallization; in step S5, the programmed drying includes pre-freezing, sublimation drying and analytical drying steps. The temperatures of pre-freezing, the sublimation drying and the analytical drying are increased in sequence.

Preferably, in step S1-1, the adsorption includes one or more of stirring adsorption and column chromatography adsorption, the adsorbent includes one or more of silica gel and alumina, and is eluted with pure ethanol. The crude paeoniflorin glycoside is obtained; in step S1-2, the volume ratio of the alkane derivative to the lower alcohol in the alkane derivative-lower alcohol mixed solvent is 3 to 7:1; in step S2, the reaction is carried out at room temperature , the reaction time is 2 to 5h; in step S4, after crystallization, it also includes the process of filtration, rinsing, and re-filtration to obtain the paeoniflorin glycoside-6'-O-benzenesulfonate fine product B; in step S5, The temperature of the pre-freezing is -60~-10°C, the temperature of the sublimation drying is -10~0°C, and the temperature of the analytical drying is 5~65°C.

In step S1-1, generally speaking, in addition to more than 20% of paeoniflorin glycosides, the crude paeoniflorin glycosides also contain a large amount of tannins, sugar gums and other impurities. If column chromatography is used directly for separation, it will not only affect The purity of paeoniflorin glycosides also has a great impact on the separation efficiency. Therefore, using adsorbents to remove impurities such as tannins and sugar gums in paeoniflorin glycosides can further improve the purity and separation efficiency of the resulting paeoniflorin glycosides.

The adsorbents used in the present invention, such as silica gel, alumina, etc., are low in price and have good separation effects, and can reduce the cost of industrial production of paeoniflorin-6'-O-benzenesulfonate. In order to further improve the separation efficiency of paeoniflorin glycosides, the crude paeoniflorin glycosides can be dissolved in a solvent consistent with the eluent used in column chromatography before column chromatography.

Preferably, in step S1-1, the mass ratio of the adsorbent to the medicinal material extract is 1-4:1, and the volume-mass ratio of pure ethanol to the adsorbent is 2-6ml:1g; step S1- In 2, before the silica gel column chromatography separation, the crude paeoniflorin derivative is dissolved in the alkane derivative-lower alcohol mixed solvent, and the alkane derivative in the alkane derivative-lower alcohol mixed solvent includes ethyl acetate. , one or more of isopropyl acetate, chloroform, and dichloromethane, and the lower alcohol in the alkane derivative-lower alcohol mixed solvent includes one or more of methanol, ethanol, and isopropyl alcohol. ; In step S3, before the silica gel column chromatography separation, use the halogenated hydrocarbon-lower alcohol mixed solvent to dissolve the paeoniflorin-6'-O-benzenesulfonate crude product; in step S4, use the Rinse with poor solvent; in step S5, the pre-freezing operation is to cool down to -60~-10°C and keep it warm for 30~300min, and the sublimation drying operation is to raise the temperature to -10~0°C within 30~180min and keep it warm for 30~180min. The temperature is maintained for 180 to 720 minutes. The analytical drying operation is to raise the temperature to 5 to 20°C within 30 to 240 minutes and keep the temperature for 60 to 600 minutes, and then raise the temperature to 50 to 65°C within 30 to 240 minutes and keep the temperature for 300 to 1440 minutes.

Preferably, in step S1-2, before the silica gel column chromatography separation, the alkane derivative-lower alcohol mixed solvent is used to dissolve the crude paeoniflorin glycoside in a volume mass ratio of 1 to 3 ml: 1 g, and the alkane derivative is The substance-lower alcohol mixed solvent is an ethyl acetate-methanol mixed solvent or an ethyl acetate-ethanol mixed solvent, and the volume ratio of the alkane derivative to the lower alcohol in the alkane derivative-lower alcohol mixed solvent is 4 to 6:1; In step S3, before the silica gel column chromatography separation, use the halogenated hydrocarbon-lower alcohol mixed solvent to dissolve the crude paeoniflorin-6'-O-benzenesulfonate ester in a volume mass ratio of 1 to 3 ml: 1 g. ; In step S5, the pre-freezing operation is to lower the temperature to -40~-10°C and keep it warm for 90~300min, and the sublimation drying operation is to raise the temperature to -10~0°C within 60~180min and keep it warm for 240~600min. , the operation of analytical drying is to raise the temperature to 5-15°C within 60-180 min and keep it warm for 90-540 min, and then raise the temperature to 50-60°C within 60-180 min and keep it warm for 480-1440 min.

A kind of paeoniflorin-6’-O-benzenesulfonate obtained by the preparation method of paeoniflorin-6’-O-benzenesulfonate has the following structure:

The obtained paeoniflorin-6'-O-benzenesulfonate has good blood-enriching effect.

Compared with the prior art, the implementation of the present invention has the following beneficial effects:

The present invention proposes for the first time a process route for preparing paeoniflorin-6'-O-benzenesulfonate. The route is simple, the conditions are mild, and it is suitable for industrial production. The synthesis method in the present invention has the advantages of high yield, small amount of catalyst and organic solvent, short reaction time, and no need to drop raw materials. In the silica gel column chromatography purification step of paeoniflorin glycosides in the present invention, the elution system of ethyl acetate-methanol or ethyl acetate-ethanol is preferably used. Compared with the elution system containing halogenated hydrocarbons, it can not only reduce the elution The dosage of remover can also improve the safety of people and industrial production. The drying process of the present invention can ensure that the solvent residue of the finished product of paeoniflorin-6'-O-benzenesulfonate is qualified without increasing the content of related substances and ensure that the paeoniflorin-6'-O-benzenesulfonate product is qualified. High quality of finished acid ester products.

Description of the drawings

Figure 1 is a hydrogen spectrum of the product of Example 1 of the present invention.

Figure 2 is a carbon spectrum of the product of Example 1 of the present invention.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the concept and technical effects of the present invention will be clearly and completely described below in conjunction with the accompanying drawings to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without exerting creative efforts are all protection scope of the present invention.

Example 1

In this example, a paeoniflorin-6'-O-benzenesulfonate is prepared. The specific process is:

A: Take 500g of white peony slices, add 4L of 95% ethanol for hot reflux extraction, filter and concentrate to obtain 31.9g of medicinal extract, then add 63.8g of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column , use 100 mL ethanol to elute, collect the eluate, and concentrate to obtain 15.4 g of crude paeoniflorin glycoside, with a purity of 25.3%. The obtained crude paeoniflorin glycoside was dissolved in 30 mL of solvent (chloroform: isopropanol = 3:1), filled with 200g of silica gel, and the eluent was chloroform: isopropanol = 5:1, and passed through a silica gel column layer The elution was analyzed, and a total of 1 L was eluted. The eluate was collected and concentrated under reduced pressure to obtain 3.1 g of paeoniflorin glycoside with a purity of 91.2%.

B: Use 12.2 mL acetone to fully dissolve the obtained paeoniflorin glycoside, add 0.061 g dimethyltin dichloride and 0.75 mL triethylamine, then add 0.775 g benzenesulfonyl chloride, and stir the reaction. After reacting for 2 hours, add 25 mL of ethyl acetate and 18.5 mL of pure water, stir for 5 min, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 3.75 g of paeoniflorin-6'-O-benzenesulfonate crude product with a purity of 84.7%. Use 10 mL of solvent (chloroform: methanol = 10:1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate and fill it with 40 g of silica gel. The eluent is chloroform: methanol = 9:1. , eluted by silica gel column chromatography and monitored by TLC, a total of 225ml of eluent was eluted, and the fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate was collected and concentrated under reduced pressure to obtain 2.65g of paeoniflorin. Glycoside-6'-O-benzenesulfonate, purity 96.3%.

C: Use 47.7 mL of isopropyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 26.5 mL of n-hexane to stir and crystallize, filter, rinse with n-hexane, and filter again to obtain 2.35 g Paeoniflorin-6'-O-benzenesulfonate with a purity of 98.4%.

D: Add 2.35g of paeoniflorin-6'-O-benzenesulfonate with a purity of 98.4% obtained in the previous step to the mixed solvent (9.4mL dimethyl carbonate, 0.59mL acetone and 1.76mL water), and stir Dissolve it completely, place it in a plate, and perform the drying procedure according to the parameters in Table 1. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.

Table 1 Example 1 drying parameters and sample quality inspection table

Taking Example 1 as an example, the hydrogen spectrum of the product is shown in Figure 1 and the carbon spectrum is shown in Figure 2. After identification, it was determined that the product had the following structure:

Example 2

A: Take 500g of white peony slices, add 600ml of 80% ethanol for thermal reflux extraction, filter and concentrate to obtain 37.6g of medicinal extract, then add 112.8g of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column , use 150 mL ethanol to elute, collect the eluate, and concentrate to obtain 17.95g of crude paeoniflorin glycoside, with a purity of 22.6%. The obtained crude paeoniflorin glycoside was dissolved in 36 mL of solvent (ethyl acetate: methanol = 4: 1), filled with 215.5 g of silica gel, and the eluent was ethyl acetate: methanol = 4: 1, and was eluted by silica gel column chromatography. , a total of 750 ml was eluted, the eluate was collected, and concentrated under reduced pressure to obtain 3.9 g of paeoniflorin glycoside with a purity of 85.4%.

B: Use 8 mL of acetonitrile to fully dissolve the obtained paeoniflorin glycoside, add 0.078g di-tert-butyltin dichloride and 1.315g sodium bicarbonate, then add 1.17g benzenesulfonyl chloride, and stir the reaction. After reacting for 2 hours, add 39 mL of ethyl acetate and 19.5 mL of pure water, stir for 5 min, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 4.15 g of paeoniflorin-6'-O-benzenesulfonate crude product with a purity of 80.6%. Use 8 mL of solvent (chloroform: ethanol = 8: 1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate, and fill it with 37.5 g of silica gel. The eluent is chloroform: ethanol = 8: 1. Elute through silica gel column chromatography and monitor by TLC. Elute a total of 290ml of eluent. Collect the fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate and concentrate under reduced pressure to obtain 2.85g of paeoniflorin. Ester glycoside-6'-O-benzenesulfonate, purity 95.2%.

C: Use 45 mL of isobutyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 28.5 mL of n-hexane to stir and crystallize, filter, rinse with n-hexane, and filter again to obtain 2.5 g of purity It is 98.1% paeoniflorin-6'-O-benzenesulfonate.

D: Add 2.5g of paeoniflorin-6'-O-benzenesulfonate with a purity of 98.1% obtained in the previous step to the mixed solvent (20mL dimethyl carbonate, 1.26mL methyl ethyl ketone and 3.76mL water), and stir Dissolve it completely, place it in a plate, and perform the drying procedure according to the parameters in Table 2. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.

Table 2 Example 2 drying parameters and sample quality inspection table

After identification, it was determined that the product had the following structure:

Example 3

A: Take 1kg of white peony slices, add 6L of absolute ethanol for thermal reflux extraction, filter and concentrate to obtain 31g of medicinal extract, then add 183g of neutral alumina for stirring and adsorption, add the adsorbed neutral alumina to the chromatography column, and use Elute with 180 mL of ethanol, collect the eluate, and concentrate to obtain 36.4 g of crude paeoniflorin glycoside, with a purity of 27.3%. The obtained crude paeoniflorin glycoside was dissolved in 72.8 mL of solvent (isopropyl acetate: methanol = 6:1), filled with 400 g of silica gel, and the eluent was isopropyl acetate: methanol = 6:1, and subjected to silica gel column chromatography. After elution, a total of 1.82L was eluted. The eluate was collected and concentrated under reduced pressure to obtain 8.86g of paeoniflorin glycoside with a purity of 87.3%.

B: Use 18 mL of dimethyl sulfoxide to fully dissolve the obtained paeoniflorin glycoside, add 0.0266 g of dimethyl tin sulfide and 1.96 g of potassium carbonate, then add 2.4 g of benzenesulfonyl chloride, and stir the reaction. After reacting for 2 hours, add 100 mL of ethyl acetate and 60 mL of pure water, stir for 5 min, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 7.2 g of paeoniflorin-6'-O-benzenesulfonate crude product with a purity of 88.2%. Use 18 mL of solvent (dichloromethane: ethanol = 10: 1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate and fill it with 57.6 g of silica gel. The eluent is dichloromethane: ethanol = 10: 1. Elute through silica gel column chromatography and monitor by TLC. Elute a total of 920ml of eluent. Collect the fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate and concentrate under reduced pressure to obtain 5.8g of paeoniflorin. Ester glycoside-6'-O-benzenesulfonate, purity 96.7%.

C: Use 69 mL of isopropyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 46 mL of isopropyl ether to stir and crystallize, filter, rinse with isopropyl ether, and filter again to obtain 5.2 g. Paeoniflorin-6'-O-benzenesulfonate with a purity of 98.2%.

D: Add 5.2g of paeoniflorin-6'-O-benzenesulfonate with a purity of 98.9% obtained in the previous step to the mixed solvent (20.8mL dimethyl carbonate, 2.6mL acetone and 2.6mL water), and stir Dissolve it completely, place it in a plate, and proceed with the drying procedure according to the parameters in Table 3. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.

Table 3 Example 3 drying parameters and sample quality inspection table

Example 4

A: Take 5kg of white peony slices, add 50L of 95% ethanol for thermal reflux extraction, filter and concentrate to obtain 401.4g of medicinal extract, then add 600g of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column. Use 1.2L ethanol to elute, collect the eluate, and concentrate to obtain 208.5g of crude paeoniflorin glycoside, with a purity of 28.4%. The obtained crude paeoniflorin glycoside was dissolved in 417 mL of solvent (methylene chloride: ethanol = 6:1), filled with 2.5 kg of silica gel, and the eluent was methylene chloride: ethanol = 6:1, and was eluted by silica gel column chromatography. , a total of 11.5L was eluted. The eluate was collected and concentrated under reduced pressure to obtain 46.25g of paeoniflorin glycoside with a purity of 93.6%.

B: Use 92.5 mL of acetonitrile to fully dissolve the obtained paeoniflorin, add 0.46 g of dimethyltin dichloride and 11.2 ml of triethylamine, then add 11.5 g of benzenesulfonyl chloride, and stir the reaction. After reacting for 2 hours, add 465 mL of ethyl acetate and 230 mL of pure water, stir for 5 min, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 39 g of paeoniflorin-6'-O-benzenesulfonate crude product with a purity of 92.5%. Use 80mL of solvent (chloroform: ethanol = 8:1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate, and fill it with 234g of silica gel. The eluent is chloroform: ethanol = 9:1. , eluted by silica gel column chromatography and monitored by TLC, a total of 2.73L of eluent was eluted, and the fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate was collected and concentrated under reduced pressure to obtain 27.5g of paeoniflorin. Ester glycoside-6'-O-benzenesulfonate, purity 96.9%.

C: Use 550mL of isopropyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 330mL of petroleum ether to stir and crystallize, filter, rinse with petroleum ether, and filter again to obtain 24g with a purity of 99.1 % of paeoniflorin-6'-O-benzenesulfonate.

D: Add 24g of paeoniflorin-6'-O-benzenesulfonate with a purity of 99.1% obtained in the previous step to the mixed solvent (57.6mL dimethyl carbonate, 3.6mL acetone and 10.8mL water), and stir until Completely dissolve, place in a plate, and perform drying procedures according to the parameters in Table 4. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.

Table 4 Example 4 drying parameters and sample quality inspection table

Example 5

A: Take 10kg of white peony slices, add 70L of 90% ethanol for thermal reflux extraction, filter and concentrate to obtain 903g of medicinal extract, then add 1.8kg of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column. Use 3.6 L of ethanol to elute, collect the eluate, and concentrate to obtain 505 g of crude paeoniflorin glycoside, with a purity of 23.6%. The obtained crude paeoniflorin glycoside was dissolved in 1000 mL of solvent (ethyl acetate: methanol = 6: 1), filled with 6.0 kg of silica gel, and the eluent was ethyl acetate: methanol = 6: 1, and was eluted by silica gel column chromatography. , a total of 29L was eluted, the eluate was collected, and concentrated under reduced pressure to obtain 153g of paeoniflorin glycoside with a purity of 87.5%.

B: Use 380 mL of acetone to fully dissolve the obtained paeoniflorin glycoside, add 4.6 g of dimethyltin dichloride and 15 ml of triethylamine, then add 42.8 g of benzenesulfonyl chloride, and stir the reaction. After reacting for 2 hours, add 1.5L ethyl acetate and 750mL pure water, stir for 5 minutes, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 162 g of paeoniflorin-6'-O-benzenesulfonate crude product with a purity of 85.2%. Use 400 mL of solvent (chloroform: methanol = 10: 1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate, and fill it with 1.8 kg of silica gel. The eluent is chloroform: methanol = 10: 1. Elute through silica gel column chromatography and monitor by TLC. A total of 11.2L of eluent is eluted. The fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate is collected and concentrated under reduced pressure to obtain 123g of paeoniflorin. Ester glycoside-6'-O-benzenesulfonate, purity 95.1%.

C: Use 1845mL of isobutyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 1353mL of n-hexane to stir and crystallize, filter, rinse with n-hexane, and filter again to obtain 106g with a purity of 98.5 % of paeoniflorin-6'-O-benzenesulfonate.

D: Add 106g of paeoniflorin-6'-O-benzenesulfonate with a purity of 98.5% obtained in the previous step to the mixed solvent (1.272L dimethyl carbonate, 79.5mL methyl ethyl ketone and 238.5mL water), and stir Dissolve it completely, place it in a plate, and perform the drying procedure according to the parameters in Table 5. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.

Table 5 Example 5 drying parameters and sample quality inspection table

Example 6

A: Take 50kg of white peony slices, add 500L of 95% ethanol for thermal reflux extraction, filter and concentrate to obtain 4.0kg of medicinal extract, then add 8.0kg of neutral alumina for stirring and adsorption, and add the adsorbed neutral alumina to the chromatography column , use 16.0L ethanol to elute, collect the eluate, and concentrate to obtain 1.8 kg of crude paeoniflorin glycoside, with a purity of 20.3%. The obtained crude paeoniflorin glycoside was dissolved in 3.6L solvent (ethyl acetate: ethanol = 6:1), filled with 20kg silica gel, and the eluent was ethyl acetate: ethanol = 6:1, and was eluted by silica gel column chromatography. , a total of 90L was eluted, the eluate was collected, and concentrated under reduced pressure to obtain 382g of paeoniflorin glycoside with a purity of 85.2%.

B: Use 900 mL acetone to fully dissolve the resulting paeoniflorin glycoside, add 10.6 g diethyl tin dichloride and 60 ml triethylamine, then add 103 g benzenesulfonyl chloride, and stir the reaction. After reacting for 2 hours, add 3.8L ethyl acetate and 1.9L pure water, stir for 5 minutes, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 402 g of paeoniflorin-6'-O-benzenesulfonate crude product with a purity of 82.8%. Use 800mL of solvent (chloroform: ethanol = 8:1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate, and fill it with 4.0kg of silica gel. The eluent is chloroform: ethanol = 8: 1. Elute through silica gel column chromatography and monitor by TLC. Elute a total of 30L of eluent. Collect the fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate and concentrate under reduced pressure to obtain 307g of paeoniflorin. Glycoside-6'-O-benzenesulfonate, purity 95.6%.

C: Use 4.0L isopropyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 2.8L isopropyl ether to stir and crystallize, filter, rinse with isopropyl ether, and filter again to obtain 286g of paeoniflorin-6'-O-benzenesulfonate with a purity of 98.4%.

D: Add 286g of paeoniflorin-6'-O-benzenesulfonate with a purity of 98.4% obtained in the previous step to the mixed solvent (1.144L dimethyl carbonate, 71.5mL acetone and 214.5mL water), and stir until Completely dissolve, place in a plate, and perform drying procedures according to the parameters in Table 6. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.

Table 6 Example 6 drying parameters and sample quality inspection table

Comparative example 1

This comparative example uses paeoniflorin glycoside (purity 98%) instead of peony extract as the starting material to prepare paeoniflorin glycoside-6'-O-benzenesulfonate, which undergoes the following processes:

Existing technology:

B: Add 6g of paeoniflorin glycoside (purity 98%) into the reaction bottle, add 240 mL (pyridine: chloroform = 1:1) mixed solution to dissolve, and then add 228 mg of 4-dimethylaminopyridine for activation for 30 minutes. Within 3 hours, dissolve 3 ml of benzene sulfonyl chloride in 240 mL of chloroform and drop it into the reaction solution for reaction. The reaction temperature is 25°C. After the dropwise addition, continue the reaction for 12 hours. After the reaction is completed, wash with water three times, 80 ml each time, and keep The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to obtain 4.9 g, with a purity of 50.6% and a yield of 32.6%.

C: Use 12 mL of solvent (chloroform: absolute ethanol = 3:1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate, and use 36 g of silica gel for column chromatography separation (eluent is tris Methyl chloride: methanol = 25:1), a total of 1 L was eluted, and 1.68 g of paeoniflorin-6'-O-benzenesulfonate was obtained, with a purity of 97.3%.

In order to more clearly see the difference between the existing preparation process and the process of the present invention, step D is omitted.

The process of the present invention:

B: Take 6g of paeoniflorin glycoside (purity 98%), use 25mL of acetone to fully dissolve it, add 0.04g of dimethyltin dichloride for 10 minutes, then add 2.5mL of triethylamine, and then add 2g of benzene Sulfonyl chloride, stir the reaction for 2 hours, and detect by TLC. After the reaction is complete, add 60 mL of ethyl acetate and 30 mL of pure water, stir for 15 min, and terminate the reaction. Leave to stand for 0.5 h, take the organic layer, and concentrate under reduced pressure to obtain 6.82 g of paeoniflorin-6'-O-benzenesulfonate crude product, with a purity of 92.6% and a yield of 83.1%. Use 13.64mL of solvent (chloroform: absolute ethanol = 3:1) to dissolve the crude paeoniflorin-6'-O-benzenesulfonate, and fill it with 54.6g of silica gel. The eluent is chloroform: ethanol. =12:1, eluted by silica gel column chromatography and monitored by TLC, a total of 410mL of eluent was eluted, and the fraction containing only paeoniflorin glycoside-6'-O-benzenesulfonate was collected and concentrated under reduced pressure to obtain 5.18 g of paeoniflorin-6'-O-benzenesulfonate, with a purity of 97.7%.

C: Use 72.5 mL of isopropyl acetate to completely dissolve the above-mentioned paeoniflorin-6'-O-benzenesulfonate, then add 51.8 mL of n-hexane to stir and crystallize, filter, rinse with n-hexane, and filter again to obtain 4.93 g. Paeoniflorin-6'-O-benzenesulfonate with a purity of 99.2%.

Comparative example 2

Steps A to C are the same as in Embodiment 4, except that step D is as follows:

D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, place it in a vacuum drying oven, and perform vacuum drying according to the parameters in Table 7. After drying, samples are collected, sampled and tested for solvent residues and related substances.

Table 7 Comparative Example 2 drying parameters and sample quality inspection table

Comparative example 3

Steps A to C are the same as in Embodiment 4, except that step D is as follows:

D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, add 20 mL of methanol and stir to dissolve completely, place it in a vacuum drying oven, and perform vacuum drying according to the parameters in Table 7. After drying, samples are collected, sampled and tested for solvent residues and related substances.

Table 8 Comparative Example 3 drying parameters and sample quality inspection table

Comparative example 4

Steps A to C are the same as in Embodiment 4, except that step D is as follows:

D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, add 20 mL of acetone and stir to dissolve completely, place it in a vacuum drying box, and perform vacuum drying according to the parameters in Table 8. After drying, samples are collected, sampled and tested for solvent residues and related substances.

Table 9 Comparative Example 4 drying parameters and sample quality inspection table

Comparative example 5

Steps A to C are the same as in Embodiment 4, except that step D is as follows:

D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, add solvent (100 mL of dimethyl carbonate), stir to completely dissolve, place in a plate, and perform drying procedures according to the parameters in Table 9. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.

Table 10 Comparative Example 5 drying parameters and sample quality inspection table

Comparative example 6

Steps A to C are the same as in Embodiment 4, except that step D is as follows:

D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, add mixed solvent (85mL dimethyl carbonate and 15mL acetone), stir to completely dissolve, place in a plate, and dry according to the parameters in Table 10 program. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.

Table 11 Comparative Example 6 drying parameters and sample quality inspection table

Comparative example 7

Steps A to C are the same as in Embodiment 4, except that step D is as follows:

D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, add mixed solvent (80mL tert-butyl alcohol + 20mL water) and stir until completely dissolved, place it in a plate, and perform the drying procedure according to the parameters in Table 11. After drying, the sample powder is collected, sampled and tested for solvent residues and related substances.

Table 12 Comparative Example 7 drying parameters and sample quality inspection table

Comparative example 8

Steps A to C are the same as in Embodiment 4, except that step D is as follows:

D: Weigh 20g of paeoniflorin-6'-O-benzenesulfonate, add 100 mL of a mixed solvent composed of dimethyl carbonate, acetone and water in different proportions according to Table 12, stir to completely dissolve, and perform the program formula Dry, collect sample powder, take samples and test for solvent residues and related substances.

Table 13 Mixed solvent status and sample quality inspection table with different proportions

Effect example 1

It can be seen from Examples 1 to 2 that in the purification step of paeoniflorin glycosides, the chloroform-alcohol system has a better separation effect, but the solvent used is high. The ethyl acetate-alcohol system is more suitable for large-scale preparation. .

It can be seen from Examples 1 to 6 that by gradually amplifying the reaction, the required dosages of reaction solvents and eluents are within the scope of the claims and have good stability.

It can be found from Comparative Example 1 that if this process directly uses paeoniflorin glycoside with a purity higher than 98% as raw material, the yield can reach more than 80%, which is much higher than the existing process, and the hydroxyl selectivity of the reaction is better. , the product has higher purity and fewer impurities than the existing process, and it is easier to prepare high-purity paeoniflorin-6'-O-benzenesulfonate through silica gel column chromatography. The column chromatography uses the eluent of chloroform: methanol = 6:1 to 12:1 to have a greater polarity than the existing process eluent (chloroform: methanol = 20:1 to 45:1). properties and stronger elution capacity. While ensuring high purity of paeoniflorin-6-O'-benzenesulfonate, it can save solvent consumption and speed up separation efficiency.

According to the provisions of the Pharmacopoeia, the residues of various solvents used in the present invention in the finished drug should basically be controlled below 0.5%. From the comparison between Example 4 and Comparative Examples 2 to 7, it can be found that using dimethyl carbonate, acetone and water, or a combination of dimethyl carbonate, methyl ethyl ketone and water as the solvent, the effect of paeoniflorin-6-O' - The benzenesulfonate ester is dissolved and then dried in a programmed manner. The solvent residue can meet the standards and there is no significant increase in related substances. The quality of the dried product is guaranteed. When methanol is used as the solvent to vacuum-dry paeoniflorin-6-O'-benzenesulfonate, although the solvent residue can meet the standards, the related substances increase significantly. In Comparative Example 4, acetone was used as the solvent to vacuum-dry paeoniflorin-6-O'-benzenesulfonate. Although there was no significant increase in related substances, the solvent residue in acetone could not be reduced below the standard. In Comparative Example 5, only dimethyl carbonate was used as the solvent, and the paeoniflorin-6-O'-benzenesulfonate was dried in a programmed manner. The dimethyl carbonate solvent residue could not be reduced below the standard. Similarly, in Comparative Example 6, a mixed solvent of dimethyl carbonate and acetone was used to perform programmed drying of paeoniflorin-6-O'-benzenesulfonate, and the solvent residue could not be reduced below the standard. Comparative Example 7 used a mixed solvent of tert-butyl alcohol and water to perform programmed drying of paeoniflorin-6-O'-benzenesulfonate. Although the solvent residue could meet the standards, related substances increased significantly. It can be seen that only by using the specific mixed solvent of the present invention can the requirements that the dissolved residues meet the standards and there is no increase in related substances can be met.

It can be seen from Comparative Example 8 that when a mixed solvent composed of dimethyl carbonate, acetone and water is used as the solvent for programmed drying, not all proportions can obtain qualified finished products. By setting mixed solvents at different proportions, the results of solvent residues and related substances in the finished product are investigated. When the acetone:water ratio in the mixed solvent is less than 1:4, as shown in groups 1, 2, and 8 in the table, the mixed system will appear stratified, and the dimethyl carbonate in the finished product will exceed the limit, affecting the drying effect; and when the mixture When the acetone content in the system exceeds 10%, as shown in Group 4, the residual acetone in the finished product exceeds the limit. When the volume ratio of dimethyl carbonate, acetone and water is 4~11:0.2~0.6:1, the mixed solvent system does not stratify, the solvent residues of dimethyl carbonate and acetone meet the specified requirements, and there is no obvious relevant substance after drying Increase. It can be seen that only by using a mixed solvent of dimethyl carbonate, C3~4 ketones and water in a specific ratio can the dissolved residues meet the standards and there is no increase in related substances.

Effect example 2

Blood enrichment experiment of paeoniflorin-6'-O-benzenesulfonate

1. After adaptive feeding, 90 mice were randomly divided into 9 groups according to body weight, half male and half female, namely control group, model group, positive drug group, paeoniflorin glycoside, and paeoniflorin glycoside-6'-O- There are three dose groups (120, 60, 30mg/kg) of benzenesulfonate, high, medium and low respectively, with 10 animals in each group. For the other seven groups except the control group, 0.5 mL of blood was bled from the fundus venous plexus and administered intragastrically 24 hours later. The positive drug group was intragastrically administered compound donkey-hide gelatin slurry at 20 ml/kg, and the control and model groups were intragastrically administered distilled water at 120 mg/kg. On

days

0, 5, and 10 after administration, 30 μL of blood was collected from the orbit to detect the number of red blood cells (see Table 14).

2. Statistical methods: The experimental data were analyzed statistically by One-way ANOVA using SPSS software, and the results were expressed as x±s.

Table 14 Effects of paeoniflorin-6'-O-benzenesulfonate and paeoniflorin on the number of red blood cells in anemia mice (x±s, n=8)

Note: Compared with day 0, *P<0.05, **P<0.01.

It can be seen from the experimental results that both paeoniflorin-6'-O-benzenesulfonate and paeoniflorin can effectively increase the number of red blood cells in mice, and the effect of paeonilide-6'-O-benzenesulfonate is greater. It is better than paeoniflorin glycoside, and the effect of high dose of paeoniflorin glycoside-6'-O-benzenesulfonate is close to that of compound donkey-hide gelatin. Compared with paeoniflorin, the reason why paeonilide-6'-O-benzenesulfonate is more effective may be due to increased lipophilicity and improved in vivo bioavailability.

What is disclosed above is only the preferred embodiment of the present invention. Of course, it cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope of the present invention.

Claims

A preparation method of paeoniflorin-6’-O-benzenesulfonate, which is characterized in that it includes the following steps:

S1: Extract paeoniflorin glycosides from medicinal materials;

S2: Add an organic metal catalyst, an acid binding agent, and benzenesulfonyl chloride to the paeoniflorin glycoside in proportion to carry out the reaction. After the reaction is terminated, the reaction product is concentrated to obtain a crude paeoniflorin glycoside-6'-O-benzenesulfonate ester;

S3: Separate the crude paeoniflorin-6'-O-benzenesulfonate product to obtain paeonilide-6'-O-benzenesulfonate fine product A;

S4: After dissolving the paeoniflorin-6'-O-benzenesulfonic acid ester boutique A in a good solvent, add a poor solvent for crystallization, and obtain paeoniflorin-6'-O-benzenesulfonic acid after crystallization. Ester Boutique B;

S5: Dissolve the paeoniflorin-6'-O-benzenesulfonate boutique B in a mixed solvent, the mixed solvent includes dimethyl carbonate, C3~4 ketones and water, and perform programmed drying. Finally, the finished product of paeoniflorin-6'-O-benzenesulfonate is obtained.
The preparation method according to claim 1, characterized in that it includes the following steps:

S1: Includes the following steps:

S1-1: The medicinal materials are extracted with alcohol, the extract is filtered and concentrated to obtain the medicinal material extract, and the crude paeoniflorin glycoside is obtained through adsorption and elution;

S1-2: The crude paeoniflorin glycoside is separated by column chromatography to obtain the paeoniflorin glycoside, and the purity of the paeoniflorin glycoside is above 80%;

S2: Add the organometallic catalyst, the acid binding agent, and the benzenesulfonyl chloride to the paeoniflorin glycoside in proportion, carry out the reaction in the reaction solvent, and then add the organic phase and the aqueous phase to terminate the reaction and perform extraction. , after concentration, the crude paeoniflorin-6'-O-benzenesulfonate ester is obtained;

S3: Separate the crude paeoniflorin-6'-O-benzenesulfonate ester through column chromatography, collect the eluent, and obtain the paeonilide-6'-O-benzenesulfonate fine product A , the purity of the paeoniflorin-6'-O-benzenesulfonate fine product A is above 95%;

S4: After dissolving the paeoniflorin-6'-O-benzenesulfonate fine product A with the good solvent, adding the poor solvent for crystallization, and obtaining paeonilide-6'-O after crystallization. - Fine benzenesulfonate ester B, the purity of the paeoniflorin-6'-O-benzenesulfonate fine ester B is above 98%;

S5: Dissolve the paeoniflorin-6'-O-benzenesulfonate boutique B in the mixed solvent, which includes dimethyl carbonate, C3~4 ketones and water in a volume ratio of 4 ~11:0.2~0.6:1 is mixed, and programmed drying is performed to finally obtain the finished product of paeoniflorin-6'-O-benzenesulfonate.
The preparation method according to claim 2, characterized in that in step S1-1, the medicinal materials include one or more of white peony root, red peony root, paeoniae root, dog head, peony root, and purple peony root, and 80% is used. ~100% ethanol or 80% ~ 100% methanol extraction, the extraction method includes one of cold soaking, hot reflux or microwave-assisted extraction; in step S1-2, the column chromatography separation is silica gel column chromatography separation, elution The agent is an alkane derivative-lower alcohol mixed solvent; in step S2, the organometallic catalyst includes C1 to C4 alkyl tin halide, aryl tin halide, C1 to C4 alkyl tin oxide, C1 to C4 alkyl One or more tin sulfides, the acid binding agent includes one of triethylamine, N, N'-diisopropylethylamine, pyridine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate. One or more, the reaction solvent includes acetonitrile, tetrahydrofuran, acetone, pyridine, methyl ethyl ketone, N,N'-dimethylformamide, 1,4-dioxane, and dimethyl sulfoxide. One or more, the organic phase is ethyl acetate; in step S3, the column chromatography separation is silica gel column chromatography, and the eluent is a halogenated hydrocarbon-lower alcohol mixed solvent; in step S4, the The good solvent includes one or more of isopropyl acetate and isobutyl acetate, and the poor solvent includes one or more of n-hexane, petroleum ether, isopropyl ether, and isooctane; in step S5, The mixed solvent includes dimethyl carbonate, C3-4 ketones and water mixed in a volume ratio of 5-8:0.3-0.5:1.
The preparation method according to claim 3, characterized in that in step S1-1, the medicinal materials include one or more of white peony root and red peony root, using 95% to 100% ethanol or 95% to 100% methanol. Extraction, the extraction method includes thermal reflux; in step S1-2, the mass ratio of the silica gel used for the silica gel column chromatography separation and the crude paeoniflorin glycoside is 10 to 20:1, and the alkane derivative-lower alcohol is mixed The volume to mass ratio of the solvent to the crude paeoniflorin glycoside is 40-65ml:1g; in step S2, the dosage of benzenesulfonyl chloride is 20%-60% of the mass of the paeoniflorin glycoside, and the organic metal The amount of catalyst used is 0.05% to 20% of the mass of the paeoniflorin glycoside, the molar ratio of the acid binding agent to the paeoniflorin glycoside is 1 to 2:1, and the reaction solvent and the paeoniflorin glycoside are The volume to mass ratio of the glycoside is 2 to 10 ml: 1 g, the volume to mass ratio of the organic phase to the paeoniflorin glycoside is 2.5 to 7.5 ml: 1 g, and the volume to mass ratio of the aqueous phase to the paeoniflorin glycoside is is 5~15ml:1g; in step S3, the mass ratio of the silica gel used for the silica gel column chromatography separation and the crude paeoniflorin-6'-O-benzenesulfonate ester is 6~12:1, the The volume to mass ratio of the halogenated hydrocarbon-lower alcohol mixed solvent and the crude paeoniflorin glycoside-6'-O-benzenesulfonate is 55-80ml:1g; in step S4, the good solvent and the paeoniflorin glycoside-6'-O-benzenesulfonate crude product are The volume-to-mass ratio of the paeoniflorin-6'-O-benzenesulfonate boutique A is 10 to 20 mL: 1 g, and the volume of the poor solvent and the paeoniflorin-6'-O-benzenesulfonate boutique A The mass ratio is 8 to 12 mL: 1 g; in step S5, the volume to mass ratio of the mixed solvent and the paeoniflorin-6'-O-benzenesulfonate fine product B is 1 to 20 mL: 1 g.
The preparation method according to claim 3, characterized in that, in step S1-1, the mass ratio of ethanol or methanol to the medicinal material is 5 to 20:1; in step S1-2, the silica gel used for silica gel column chromatography separation The mass ratio to the crude paeoniflorin glycoside is 10 to 15:1, and the volume to mass ratio of the alkane derivative-lower alcohol mixed solvent to the crude paeoniflorin glycoside is 50 to 60 ml: 1g; in step S2 , the dosage of benzenesulfonyl chloride is 25% to 30% of the mass of the paeoniflorin glycoside, the dosage of the organometallic catalyst is 0.5% to 5.0% of the quality of the paeoniflorin glycoside, and the acid binding agent The molar ratio to the paeoniflorin glycoside is 1.2-1.6:1; in step S5, the volume to mass ratio of the mixed solvent to the paeoniflorin glycoside-6'-O-benzenesulfonate fine product B is 1 ~15mL: 1g.
The preparation method according to claim 3, characterized in that, in step S1-1, the mass ratio of ethanol or methanol to the medicinal material is 5 to 10:1; in step S1-2, the silica gel used for silica gel column chromatography separation The mesh number is 60~400 mesh, and the aperture is
The volume ratio of the alkane derivative to the lower alcohol in the alkane derivative-lower alcohol mixed solvent is 1 to 10:1; in step S2, the amount of the organometallic catalyst is 0.6% to 0.6% of the mass of the paeoniflorin glycosides. 3.0%; in step S3, the mesh number of the silica gel used for the silica gel column chromatography separation is 60 to 400 mesh, and the volume ratio of the halogenated hydrocarbon to the lower alcohol in the halogenated hydrocarbon-lower alcohol mixed solvent is 6 to 12: 1; In step S4, after dissolving the paeoniflorin-6'-O-benzenesulfonate fine product A with the good solvent at 50 to 60°C, the temperature is lowered to 10 to 20°C and the poor solvent is added. Crystallization; in step S5, the programmed drying includes the steps of pre-freezing, sublimation drying and analytical drying, and the temperatures of the pre-freezing, sublimation drying and analytical drying increase in sequence.
The preparation method according to claim 6, characterized in that in step S1-1, the adsorption includes one or more of stirring adsorption and column chromatography adsorption, and the adsorbent includes one or more of silica gel and alumina. A variety of methods are used to elute with pure ethanol to obtain the crude paeoniflorin glycoside; in step S1-2, the volume ratio of the alkane derivative to the lower alcohol in the alkane derivative-lower alcohol mixed solvent is 3 to 7: 1; In step S2, the reaction is carried out at room temperature, and the reaction time is 2 to 5 hours; in step S4, after crystallization, it also includes the process of filtration, rinsing, and re-filtration to obtain the paeoniflorin-6'-O-benzene. Sulfonate ester boutique B; in step S5, the pre-freezing temperature is -60~-10°C, the sublimation drying temperature is -10~0°C, and the analytical drying temperature is 5~65°C.
The preparation method according to claim 6, wherein in step S1-1, the mass ratio of the adsorbent to the medicinal material extract is 1 to 4:1, and the volume to mass ratio of pure ethanol to the adsorbent is 2 to 6 ml: 1 g; in step S1-2, before the silica gel column chromatography separation, use the alkane derivative-lower alcohol mixed solvent to dissolve the crude paeoniflorin glycoside, and the alkane derivative-lower alcohol mixed solvent Alkane derivatives in the solvent include one or more of ethyl acetate, isopropyl acetate, chloroform, and methylene chloride, and lower alcohols in the alkane derivative-lower alcohol mixed solvent include methanol, ethanol, One or more isopropyl alcohols; in step S3, before the silica gel column chromatography separation, use the halogenated hydrocarbon-lower alcohol mixed solvent to dissolve the paeoniflorin-6'-O-benzenesulfonic acid The crude ester product; in step S4, rinse with the poor solvent; in step S5, the pre-freezing operation is to cool down to -60~-10°C and keep it warm for 30~300min, and the sublimation drying operation is to Raise the temperature to -10~0°C within 180min and keep it warm for 180~720min. The operation of analytical drying is to raise the temperature to 5~20°C within 30~240min and keep it warm for 60~600min, and then raise the temperature to 50~65°C within 30~240min. ℃ and keep warm for 300~1440min.
The preparation method according to claim 6, characterized in that, in step S1-2, before the silica gel column chromatography separation, the alkane derivative-lower alcohol mixed solvent is used to dissolve the alkane derivative-lower alcohol mixed solvent at a volume mass ratio of 1 to 3 ml: 1 g. Paeoniflorin crude product, the alkane derivative-lower alcohol mixed solvent is an ethyl acetate-methanol mixed solvent or an ethyl acetate-ethanol mixed solvent, and the alkane derivative and lower alcohol in the alkane derivative-lower alcohol mixed solvent are The volume ratio is 4 to 6:1; in step S3, before the silica gel column chromatography separation, use the halogenated hydrocarbon-lower alcohol mixed solvent to dissolve the paeoniflorin- 6'-O-benzenesulfonate crude product; in step S5, the pre-freezing operation is to cool down to -40~-10°C and keep the temperature for 90~300min, and the sublimation drying operation is to heat up to -10~0°C and kept at 240~600min. The analytical drying operation is to raise the temperature to 5~15°C within 60~180min and keep it at 90~540min, and then raise the temperature to 50~60°C within 60~180min and keep it at 480°C. ~1440min.
A kind of Paeoniflorin-6'-O-benzenesulfonate obtained by the preparation method of Paeoniflorin-6'-O-benzenesulfonate according to claim 1, is characterized in that it has the following structure: