Debenzoylpaeoniflorin-6'-O-benzenesulfonate, preparation method and use thereof

Abstract

The present invention relates to the technical field of pharmaceutical chemistry, and in particular to debenzoylated paeoniflorin-6'-O-benzenesulfonate, a preparation method thereof, and a use for treating Sjögren's syndrome and rheumatoid arthritis. The preparation method is to use paeoniflorin-6'-O-benzenesulfonate as a raw material, hydrolyze, extract, column chromatography and prepare liquid phase separation to obtain the debenzoylated paeoniflorin-6'-O-benzenesulfonate. The debenzoylated paeoniflorin-6'-O-benzenesulfonate of the present invention has a higher bioavailability than paeoniflorin and paeoniflorin-6'-O-benzenesulfonate, has a significant therapeutic effect on Sjögren's syndrome and rheumatoid arthritis, and can be prepared into a medicine for treating Sjögren's syndrome and rheumatoid arthritis.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to debenzoylpaeoniflorin-6'-O-benzenesulfonate, and a preparation method and use thereof. Background Art

[0002] Autoimmune diseases are a type of disease caused by pathological immune responses in the body, which damages tissues or organs and leads to organ or system dysfunction. Such diseases can affect multiple organs or tissues such as glands, joints and soft tissues, bones, muscles, and skin. Autoimmune diseases can be divided into two categories: those in which pathological damage and dysfunction are limited to independent organs targeted by antibodies or sensitized lymphocytes are called organ-specific autoimmune diseases; those in which multiple organs are damaged are called systemic autoimmune diseases. There are many types of systemic autoimmune diseases, including Sjögren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, urticaria, etc. Among them, Sjögren's syndrome and rheumatoid arthritis are relatively common systemic autoimmune diseases.

[0003] Sjögren's syndrome Sjogren's Syndrome (SS) is a chronic inflammatory autoimmune disease that invades exocrine glands, especially salivary glands and lacrimal glands. There are more than 8 million patients with this disease in my country. Currently, there is no ideal treatment for SS. Related drugs such as pilocarpine and cevimeline are mainly symptomatic treatments, aiming to control dryness symptoms and slow down the progression of the disease. However, the efficacy of such drugs is heavily dependent on the residual gland function and is accompanied by obvious adverse reactions.

[0004] Rheumatoid arthritis (RA) is a common chronic autoimmune disease characterized by erosive arthritis, and its pathological basis is synovial inflammation. The prominent features of the disease are synovial vascular hyperplasia and inflammatory cell infiltration. As the disease progresses, the synovium and cartilage are gradually destroyed, eventually leading to joint ankylosis, deformity and loss of function. Patients may become disabled, and their health and quality of life are seriously affected.

[0005] The treatment of autoimmune diseases mainly includes symptomatic treatment and causal treatment. Symptomatic treatment can effectively control symptoms, but it is easy to relapse after stopping the drug, and it cannot achieve a fundamental therapeutic effect. Therefore, it can be seen that drugs that play a fundamental therapeutic role by regulating the immune system are currently the top priority for the development of drugs for related diseases.

[0006] Total glucosides of white peony are extracted from the traditional Chinese medicine white peony root and are reported to have a certain effect on Sjögren's syndrome, but their onset of action is slow and the individual differences in efficacy are large. Previous studies have shown that a benzenesulfonated peony glycoside derivative CP-25 obtained by structurally modifying the main component of total glucosides of white peony, paeoniflorin, has a good effect on Sjögren's syndrome and is significantly better than paeoniflorin. However, CP-25 needs to be administered for a long time before it can show the effect of relieving dryness symptoms. This phenomenon may be related to its special properties. CP-25 has low solubility, slow dissolution rate, and the property of becoming sticky and forming a colloid when it comes into contact with water, which is not conducive to dispersion and absorption in the body, nor is it conducive to the development of molded preparations. Therefore, developing a compound with definite efficacy and good solubility is an important way to further develop paeoniflorin and improve its efficacy. Summary of the invention

[0007] The object of the present invention is to provide a debenzoylated paeoniflorin-6'-O-benzenesulfonate, a preparation method and an application thereof, especially an application related to the treatment of Sjögren's syndrome and rheumatoid arthritis.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] Debenzoylpeonidin-6'-O-benzenesulfonate has the following chemical structure:

[0010]

[0011] After oral administration of paeoniflorin, multi-stage metabolic reactions occur in vivo, and the products in vivo are complex. Among them, the structure obtained by debenzoylation after paeoniflorin hydrolysis is one of the main metabolites in the body, suggesting that this structure may be the main form of paeoniflorin to exert its pharmacological effect. Nevertheless, debenzoyl paeoniflorin has poor lipophilicity and is difficult to be absorbed by gastrointestinal epithelial cells, suggesting that direct administration in this form cannot achieve the purpose of improving bioavailability. Based on this, the present invention synthesizes a novel compound obtained by structural modification of paeoniflorin, debenzoyl paeoniflorin-6'-O-benzenesulfonate (CP25M2).

[0012] CP25M2 can significantly increase the amount of saliva in SS model mice and improve the degree of joint lesions in RA model animals. This product significantly inhibits lymphocyte infiltration in glandular organs by regulating immune cell function and cytokine and chemokine levels, significantly increases glandular secretion levels, and reduces joint damage. Its efficacy mechanism is based on causal treatment based on its regulation of immune function, thereby significantly improving SS and RA symptoms.

[0013] A method for preparing the debenzoylated paeoniflorin-6'-O-benzenesulfonate as described above comprises the following steps:

[0014] A. Peony glycoside-6'-O-benzenesulfonate (CP-25) was dissolved in an alkaline solvent and stirred until TLC showed that CP-25 had been completely consumed, and the reaction was completed;

[0015] B. After the reaction is completed, the system is adjusted to neutral or acidic, and the solvent is removed under reduced pressure to obtain crude product-Ⅰ;

[0016] C. Dissolving the crude product-Ⅰ in water and then extracting with an organic solvent to take the aqueous phase;

[0017] D. Concentrating the aqueous phase under reduced pressure, performing column chromatography or preparative liquid separation, collecting the eluate, and removing the solvent under reduced pressure to obtain the debenzoylpaeoniflorin-6'-O-benzenesulfonate.

[0018] During the hydrolysis reaction of step A, samples are taken at different time points for TLC detection, for example, samples are taken at 1, 2, 3, 4, 6, 8, 24, and 48 hours of the reaction, and the reaction is stopped when TLC shows that CP-25 is completely consumed. If the reaction time is too long, further side reactions will occur to generate impurities with polarity greater than or less than the target product, increasing the difficulty of separation and purification, so it is not suitable to continue the reaction.

[0019] The main product of the hydrolysis reaction is soluble in water. The main impurities in the crude product-Ⅰ are benzoic acid or sodium benzoate or a mixture of the two. The impurities can be removed to a certain extent by extraction with an organic solvent in step C.

[0020] Preferably, in step A, the alkaline solvent is 1 to 100 times the weight of paeoniflorin-6'-O-benzenesulfonate, the pH of the system is adjusted to 10 to 12, the stirring reaction temperature is room temperature to 60°C, the reaction environment is -0.1 MPa to normal pressure, and the reaction time is 15 min to 48 h.

[0021] Preferably, in step A, the alkaline solvent is 50 times the weight of paeoniflorin-6'-O-benzenesulfonate, the stirring reaction temperature is room temperature, the reaction environment is normal pressure, and the reaction time is 48 hours.

[0022] Preferably, in step A, the alkaline solvent is 50 times the weight of paeoniflorin-6'-O-benzenesulfonate, the stirring reaction temperature is 45° C., the reaction environment pressure is not higher than -0.08 MPa during the reaction, and the reaction time is 2 h.

[0023] During the reaction, air may have an adverse effect on the product, resulting in an increase in by-products (impurities). Especially when the reaction is carried out in an alcohol-containing system, the impurities are more obvious. Vacuuming can reduce the risk of product oxidation during the reaction, and even if the reaction temperature is increased to a certain extent, it will not produce more impurities, which is conducive to promoting the reaction to proceed in the forward direction.

[0024] Preferably, in step A, the alkali source in the alkaline solvent includes one or more of sodium hydroxide, potassium hydroxide, sodium methoxide, potassium carbonate, diethylamine, triethylamine, and ammonia water, and the solvent includes one or more of methanol, ethanol, isopropanol, acetone, ethyl acetate, and water; in step B, one or more of hydrochloric acid and sulfuric acid are added dropwise to adjust the system to neutrality, and the method for removing the solvent includes reduced pressure concentration, freeze drying, and spray drying; in step C, the organic solvent includes one or more of ether, petroleum ether, ethyl acetate, chloroform, and dichloromethane; in step D, the method for concentrating the aqueous phase includes reduced pressure concentration, the filler used for column chromatography or preparative liquid separation includes one of silica gel, ODS, and macroporous resin, the eluent used includes one or more of methanol, ethanol, isopropanol, acetonitrile, water, chloroform, and dichloromethane, and the method for removing the solvent includes reduced pressure concentration, freeze drying, and spray drying.

[0025] Preferably, in step A, the alkali source in the alkaline solvent is sodium hydroxide, and the solvent is methanol or water; in step A, when the alkaline solvent contains water, paeoniflorin-6'-O-benzenesulfonate is first dissolved in a non-aqueous solvent and then mixed with the alkaline solvent; in step B, 0.1-1M hydrochloric acid is added dropwise to adjust the system to neutral or acidic; in step C, the organic solvent is ether or chloroform; in step D, the aqueous phase is first adjusted to neutral.

[0026] CP-25 has special properties. It becomes sticky when it comes into contact with water and has poor solubility in solvents with high water content (when 0.1M sodium hydroxide aqueous solution is used as the solvent, the solvent multiple needs to be increased to about 50 times to achieve a good dissolution effect. If the pH of the ethanol solvent is adjusted to 12 with sodium hydroxide aqueous solution in advance, after adding CP-25 powder, a viscous substance will be formed in the case of 10 times solvent, resulting in inability to fully dissolve. Therefore, when the alkaline solvent contains water, it is necessary to first dissolve paeoniflorin-6'-O-benzenesulfonate in a non-aqueous solvent and then mix it with the alkaline solvent, otherwise the required solvent multiple is relatively large.

[0027] In step B, other commonly used acidic agents such as sulfuric acid may also be used to adjust the pH, but solid impurities such as sodium sulfate may be produced, causing trouble for subsequent operations. It is preferred to use hydrochloric acid to adjust the pH.

[0028] The system can be adjusted to acidic in step B and then adjusted to neutral in step D, or the system can be adjusted to neutral directly in step B. If the system is adjusted to neutral in step B, the main byproduct in the system is sodium benzoate soluble in water, which must be gradually removed by column chromatography. If the system is adjusted to acidic in step B, more sodium benzoate can be converted into benzoic acid that is poorly soluble in water, and some impurities can be removed after filtration, which is conducive to improving the refining efficiency.

[0029] Preferably, in step D, the filler used for column chromatography or preparative liquid separation is silica gel, and the eluent used is chloroform:methanol=15-10:1.

[0030] Preferably, in step D, the filler used for column chromatography or preparative liquid separation is ODS or macroporous resin, and the eluent used is 10% to 30% acetonitrile-water solution.

[0031] After the solvent is removed, the sample should be reconstituted with an eluent before being refined and separated.

[0032] Experiments have found that in the crude CP25M2 prepared by the method of the present invention, there may be an impurity whose retention time is only 1 min different from that of the target product CP25M2, which is extremely difficult to separate. Through continuous experiments, it has been found that the use of silica gel or ODS and macroporous resin has different separation and purification effects. The target product CP25M2 has good solubility in methanol and water, and the silica gel filler is not water-resistant, so methanol and organic solvents should be combined to form an eluent system. The experiment finally determined that when chloroform-methanol is used as the eluent, the use of silica gel column chromatography has a faster purification rate, but the impurity is eluted together with the target product CP25M2, which is suitable for the production of products with large dosage and low purity requirements. The experiment also found that the use of preparative liquid phase with ODS or macroporous resin, and the use of acetonitrile-water combination to form an eluent system can better separate the impurity from the target product CP25M2, but it takes a long time and is suitable for products with high purity requirements.

[0033] An application of the debenzoylated paeoniflorin-6'-O-benzenesulfonate in the preparation of a drug for treating autoimmune diseases.

[0034] Preferably, the autoimmune disease includes one or more of Sjögren's syndrome and rheumatoid arthritis.

[0035] The debenzoylpeonidin-6'-O-benzenesulfonate of the present invention is mixed with conventional pharmaceutically acceptable pharmaceutical excipients to prepare a pharmaceutically acceptable drug form.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The product of the present invention has the characteristics of definite efficacy and good safety for autoimmune diseases, especially Sjögren's syndrome and rheumatoid arthritis, and has a bioavailability significantly higher than that of paeoniflorin and CP-25, and can effectively solve the shortcomings of poor absorption and long onset time of total paeony glucosides in clinical use.

[0038] The technical route of the invented preparation method has mild conditions, involves excellent accessibility of materials, does not require the use of catalysts, and is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 HPLC chromatogram of debenzoylpaeoniflorin-6'-O-benzenesulfonate prepared in Example 1 of the present invention;

[0040] Figure 2 This is the HPLC chromatogram of debenzoylpaeoniflorin-6'-O-benzenesulfonate prepared in Example 6 of the present invention. DETAILED DESCRIPTION

[0041] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0042] Example 1

[0043] (1) Weigh 0.202 g of CP-25 powder into a 50 mL round-bottom flask;

[0044] (2) Add 19 mL of methanol and stir until the solid is completely dissolved;

[0045] (3) Add 1 mL of 10 M sodium hydroxide aqueous solution, at which time the pH of the reaction system is about 12;

[0046] (4) Stirring at room temperature and atmospheric pressure for 3 h, adding 1 M hydrochloric acid dropwise to adjust the pH of the reaction system to about 6-7;

[0047] (5) Filter, take the filtrate, remove the solvent under reduced pressure, separate by silica gel column chromatography, eluent is dichloromethane:methanol 15:1, concentrate under reduced pressure to obtain a white solid;

[0048] (6) The solid was dissolved in methanol and separated by preparative liquid phase. The preparative liquid phase filler was ODS and the eluent was 30% acetonitrile aqueous solution. The solvent was removed from the eluent under reduced pressure to obtain a white solid.

[0049] The obtained white solid was subjected to NMR and mass spectrometry detection, and the data are as follows:

[0050] 1H NMR(500MHz,Methanol-d4)δ7.94(d,J=8.3Hz,2H,Benzene),7.77(t,J=7.5Hz,1H,Benzene),7.67(t,J=7.8Hz,2H,Benzene) ene),5.31(s,1H,7-H),4.53(d,J=7.7Hz,1H,Glc-1'-H),4.34(dd,J=10.6,2.0Hz,1H,Glc-6'-H),4.17–4.09(m,1H,Glc- 6'-H),3.51–3.46(m,1H,Glc-5'-H),3.41(s,3H,Glc-2',3',4'-H),3.23–3.18(m,2H,9-CH2),2.62(dd,J=6.8,1.6Hz,1H ,3-H),2.34(dd,J=10.9,6.9Hz,1H,2-H),2.07–1.96(m,2H,2-H,5-H),1.71(d,J=10.8Hz,1H,5-H),1.26(s,3H,10-CH3).

[0051] 13 C NMR(126MHz,Methanol-d4)δ136.04–127.52(Benzene),108.08(4-C),100.97(Glc-1'-C),98.38(7-C),87.84(1-C),85.71( 6-C),79.47–69.64(Glc-2',3',4'5',6'-C),57.47(8-C),50.12(9-C),41.00(3-C),39.13(5-C),21.75(2-C),18.28(10-C).

[0052] ESI-HRMS(m / z):[M+Na] + Calculate for C 22 H 28 NaO 12 S + ,539.1194;found539.1190.

[0053] It can be clearly seen from the NMR and mass spectrometry data that the white solid is debenzoylpeonidin-6'-O-benzenesulfonate, as shown below:

[0054]

[0055] The obtained white solid was subjected to HPLC chromatography. The results were as follows Figure 1 shown.

[0056] Example 2

[0057] (1) Weigh 1.016 g of CP-25 powder into a 100 mL round-bottom flask;

[0058] (2) injecting 50 mL of 0.1 M sodium hydroxide aqueous solution, at which time the pH of the reaction system is about 12;

[0059] (3) After stirring at room temperature and normal pressure for 48 h, 1 M hydrochloric acid was added dropwise to adjust the pH of the reaction system to about 6-7;

[0060] (4) Filter, take the filtrate, remove the solvent under reduced pressure until the reaction solution remains about 20 mL, and extract twice with equal volumes of ether;

[0061] (5) The aqueous phase was concentrated under reduced pressure and ODS was separated using a 30% acetonitrile-water solution as the eluent. The mixture was concentrated under reduced pressure to obtain a white solid. HPLC showed that the retention time of the product was basically the same as that in Example 1.

[0062] Example 3

[0063] (1) Weigh 9.998 g of CP-25 powder into a 1000 mL three-necked round-bottom flask;

[0064] (2) injecting 500 mL of 1% sodium hydroxide methanol solution, at which point the pH of the reaction system is about 10;

[0065] (3) 45°C, vacuum stirring for 2 h, the pressure during stirring was not higher than -0.08 MPa, and after the reaction was completed, 1 M hydrochloric acid was added dropwise to adjust the pH of the reaction system to about 6-7;

[0066] (4) Filter, take the filtrate, remove the solvent under reduced pressure, add 20 mL of pure water to dissolve the product, and extract twice with equal volumes of ether;

[0067] (5) The aqueous phase was concentrated under reduced pressure, and ODS was separated using a 30% acetonitrile-water solution as the eluent. The phase was concentrated under reduced pressure to obtain a white solid. HPLC showed that the retention time of the product was basically the same as that in Example 1.

[0068] Example 4

[0069] (1) Weigh 1.905 g of CP-25 powder into a 50 mL round-bottom flask;

[0070] (2) injecting 10 mL of 10% sodium methoxide methanol solution, at which point the pH of the reaction system is about 13;

[0071] (3) After stirring at room temperature and normal pressure for 0.5 h, 1 M hydrochloric acid was added dropwise to adjust the pH of the reaction system to about 6-7;

[0072] (4) Filter, take the filtrate, remove the solvent under reduced pressure, separate by silica gel column chromatography, the eluent is dichloromethane: anhydrous ethanol 20:1, and the eluate is concentrated under reduced pressure to obtain a white solid. HPLC shows that the retention time of the product is basically the same as that in Example 1.

[0073] Example 5

[0074] (1) Weigh 1.986 g of CP-25 powder into a 50 mL round-bottom flask;

[0075] (2) Inject 9 mL of 95% ethanol solution and stir until the solid is completely dissolved;

[0076] (3) Add 1 mL of 10 M sodium hydroxide aqueous solution, at which time the pH of the reaction system is about 12;

[0077] (4) After stirring at room temperature and normal pressure for 4 hours, 1M hydrochloric acid was added dropwise to adjust the pH of the reaction system to about 6-7;

[0078] (5) Filter, take the filtrate, remove the solvent under reduced pressure, separate by silica gel column chromatography, the eluent is dichloromethane: anhydrous ethanol 20:1, and the eluate is concentrated under reduced pressure to obtain a white solid. HPLC shows that the retention time of the product is basically the same as that in Example 1.

[0079] Example 6

[0080] (1) Weigh 6.050 g of CP-25 powder into a 500 mL round-bottom flask;

[0081] (2) inject 300 mL of 0.1 M sodium hydroxide aqueous solution and stir until the solid is completely dissolved;

[0082] (3) After stirring at room temperature and pressure for 1.5 h, 1 M hydrochloric acid was added dropwise to adjust the pH of the reaction system to about 1;

[0083] (4) Stir in an ice-water bath for 30 min and let stand at 4°C overnight;

[0084] (5) Filter, take the filtrate, and extract with chloroform until no obvious benzoic acid spots are found in the aqueous phase by TLC;

[0085] (6) taking the aqueous phase, adding sodium hydroxide aqueous solution dropwise to adjust the system to neutrality;

[0086] (7) removing the solvent under reduced pressure, re-dissolving in acetone, filtering, and collecting the filtrate;

[0087] (8) The solvent was removed under reduced pressure, and the mixture was re-dissolved in 10% acetonitrile aqueous solution, and separated by ODS chromatography. The product was eluted with 10% acetonitrile aqueous solution until no obvious spots were found on TLC, and then eluted with 15% acetonitrile aqueous solution. The eluate was concentrated under reduced pressure to obtain a white solid. The HPLC chromatography detection conditions were improved. The results were as follows: Figure 2 shown.

[0088] Through NMR and mass spectrometry, the white solid was determined to be debenzoylpeonidin-6'-O-benzenesulfonate, which has the following structure:

[0089]

[0090] In this embodiment, after the reaction is completed, the pH is adjusted to acidic, which can promote the conversion of the reaction by-product sodium benzoate into benzoic acid which is insoluble in water. After standing overnight, solid benzoic acid is seen to precipitate. After filtration, a part of the benzoic acid impurities can be removed in advance. Combined with the extraction in step 5, the benzoic acid impurities can be completely removed, and there is no need to remove impurities through column chromatography, which can reduce the difficulty of subsequent column chromatography separation and save the time required for column chromatography separation.

[0091] from Figure 2 Combined with Example 1 Figure 1 It can be seen that: Figure 1 The retention time of the intermediate product and the impurity benzoic acid is very close, with a difference of only 1 minute (the product is about 21 minutes, and the benzoic acid is about 22 minutes), which makes it easy to be confused with the impurity. After the preparation and detection methods are improved, the product can be clearly separated from the impurity (the product is about 18 minutes, and the benzoic acid is about 22 minutes).

[0092] Example 7

[0093] Solubility determination: Take CP25M2 and CP-25 powders respectively, use pure water as solvent, and determine the solubility of the compound in water according to the solubility test method in the Chinese Pharmacopoeia. The results are shown in Table 1:

[0094] Table 1. Solubility determination

[0095] CP25M2 CP-25 Solubility in pure water at room temperature >100 mg / mL 2.6mg / mL

[0096] As shown in Table 1, the solubility of CP-25 is low, at the "slightly soluble" level described in the Chinese Pharmacopoeia. After structural modification, the solubility is significantly improved, reaching the "easily soluble" level described in the Chinese Pharmacopoeia. In addition, CP-25 dissolves slowly, and the instantaneous solubility of the solute added is only 0.52 mg / mL, and it can only reach the level of 2.6 mg / mL after 24 hours. In contrast, when CP25M2 is added to pure water, the solid dissolves rapidly to form a clear solution, indicating that the solubility and dissolution rate of CP25M2 are significantly improved compared with CP-25.

[0097] Solubility and dissolution rate are of great significance in the design, preparation and quality control of drug preparations. After oral administration of solid drug preparations, the drug needs to undergo a series of processes such as disintegration, dispersion, and dissolution, and diffuse to the surface of small intestinal epithelial cells in a solution state and be absorbed. On the one hand, after a drug with too low solubility or too slow dissolution rate forms a solid preparation, it is not conducive to disintegration in the body after oral administration. When the oral blood drug concentration is insufficient, it is also not conducive to the preparation of other dosage forms such as oral liquid and injection. It is difficult to solve the problem of disintegration and dispersion by adjusting the dosage form, which restricts the formulation of the compound. On the other hand, solubility and dissolution rate are important factors affecting the absorption and bioavailability of drugs in the body. Drugs with good solubility are easier to disperse and dissolve after disintegration, and are absorbed faster. The time that the drug stays in the absorption site of the small intestine after oral administration is limited, generally staying for 6-8 hours. If the drug dissolves too slowly, it may be transported to the non-absorption site in a solid form with intestinal peristalsis without complete dissolution, resulting in insufficient drug absorption. It can be seen from this that solubility and dissolution rate are important factors in drug absorption, which can affect the degree of drug absorption and thus affect the efficacy. The compound CP25M2 described in the present invention is not only easily soluble, but also dissolves rapidly, and there is no residual solid in the compound in the solvent. Compared with CP-25, it has a very significant solubility advantage and shows a broader space for formulation development.

[0098] Example 8

[0099] Evaluation of the efficacy of CP25M2 on antigen-induced Sjögren's syndrome

[0100] Preparation of antigen solution: Take C57BL / 6 mice, separate the submandibular gland, homogenize, centrifuge, discard the upper lipid layer, take the supernatant, and store it at -80℃ after aliquoting. When using, add complete Freund's adjuvant or incomplete Freund's adjuvant in a 1:1 ratio to prepare CFA-antigen solution or IFA-antigen solution, and inject it immediately after ultrasonic emulsification in an ice water bath.

[0101] Model replication: 8-week-old C57BL / 6 mice that passed quarantine were taken. After measuring the weight of the mice, the C57BL / 6 mice were randomly divided into a blank group and a model group. After grouping, on days 0 and 7 of the model group, the backs of the mice in each group were disinfected with alcohol and the antigens were injected at multiple points on the back and the base of the tail. Each mouse was injected with 0.1 mL of a 2.5 mg / ml CFA-antigen solution per 20 g of body weight. On the 14th day, the IFA-antigen solution was injected into the back and the base of the tail of the mice in the same way. Another C57BL / 6 mouse of the same age was taken as a blank control, and the blank control group was injected with normal saline. Saliva secretion was measured 6 weeks after modeling, and secondary grouping was performed after the pathological model was successfully replicated.

[0102] Saliva secretion measurement: the test was performed once a week before the mice were grouped and after the start of drug administration. The mice were anesthetized before the test. They were anesthetized by intramuscular injection of 25 mg / ml "Sule" at a volume of 0.02 ml / 10 g. Then, 0.0125 mg / ml pilocarpine saline solution was injected intraperitoneally at a volume of 0.1 ml / 10 g. After 5 minutes of injection, saliva was collected from each mouse for 10 minutes at room temperature: a cotton ball with a weighed dry weight (the dry weight range was controlled at 0.3-0.5 g) was placed in the animal's cheek, taken out after 10 minutes, and the wet weight was weighed on an electronic balance.

[0103] Mice with obvious dryness symptoms were selected and randomly and balancedly divided into a model group, a positive control group, and high-dose and low-dose groups of CP-25 and CP25M2. The positive control group was intragastrically administered with hydroxychloroquine at 70 mg / kg. The high-dose and low-dose groups of CP-25 and CP25M2 were intragastrically administered with CP-25 or CP25M2 prepared in Example 6 at 70 mg / kg and 17.5 mg / kg, respectively. The blank and model control groups were given an equal amount of solvent. Each group was administered once a day, and the saliva secretion was detected every 7 days from the date of administration. The experiment was carried out according to the sex of the animals. The results are shown in Tables 2 and 3.

[0104] Table 2. Effect of CP25M2 on saliva volume (mg) of female mice with induced Sjögren's syndrome model n=10)

[0105] 0WEEK 1WEEK 2WEEK 3WEEK Blank control group 41.263±12.713 42.831±9.108 49.388±6.140 57.168±10.035 Model control group 30.035±3.258* 24.127±8.201** 25.417±12.637*** 30.776±10.551*** Positive control group 29.617±2.816 23.851±11.411 32.843±11.688 48.336±17.12^ CP25M2 low dose group 29.19±2.092 33.19±18.143 47.92±23.957^ 41.553±8.531^ CP25M2 high dose group 30.022±3.532 32.406±13.585 39.292±10.155^ 47.705±10.270^^ CP-25 low dose group 32.513±4.638 29.172±6.579 46.357±10.755^^ 51.62±18.296^ CP-25 high dose group 29.223±2.368 34.955±14.942 44.38±13.690^ 47.392±14.725^

[0106] Note: *P<0.05, **P<0.01, ***P<0.001 vs blank control group; ^P<0.05, ^^P<0.01 vs model control group

[0107] As shown in Table 2, CP25M2 has a significant therapeutic effect on Sjögren's syndrome in female mice, can effectively increase the saliva secretion of model animals, improve dryness symptoms, and can show an improvement trend in saliva secretion after 1 week of administration. After 2 weeks of administration, the saliva secretion of animals is significantly better than that of the model control group. In contrast, the positive drug hydroxychloroquine did not show any saliva improvement trend after 1 week of administration. After 2 weeks of administration, the saliva secretion of model animals was similar to that of CP25M2 after 1 week of administration, and it took 3 weeks of administration to show a statistical difference compared with the model group, indicating that CP25M2 acts more quickly and can achieve the effect of improving saliva secretion faster than the positive drug hydroxychloroquine.

[0108] Table 3. Effect of CP25M2 on saliva volume (mg) in male induced Sjögren's syndrome model mice ( n=10)

[0109] 0WEEK 1WEEK 2WEEK 3WEEK Blank control group 53.537±3.713 49.951±10.118 52.648±5.986 54.75±5.975 Model control group 42.1475±11.399** 35.06±14.516** 33.93±9.534*** 29.791±8.694*** Positive control group 42.285±11.587 43.921±9.014 35.538±16.413 47.669±9.498^^^ CP25M2 low dose group 41.97±12.290 41.178±16.127 49.15±12.979^ 42.192±12.104^ CP25M2 high dose group 42.187±10.964 38.868±8.057 44.325±7.285^ 51.511±19.227^ CP-25 low dose group 41.472±12.968 33.955±13.537 40.966±7.900 45.297±11.529^ CP-25 high dose group 41.036±11.848 32.51±9.584 46.977±10.271^ 43.311±11.865^

[0110] Note: *P<0.05, **P<0.01, ***P<0.001 vs blank control group; ^P<0.05, ^^P<0.01, ^^^P<0.001 vs model control group

[0111] As shown in Table 3, CP25M2 has a significant therapeutic effect on Sjögren's syndrome in male mice, can effectively increase the saliva secretion of model animals, and improve dryness symptoms. After 2 weeks of administration, the saliva secretion of animals was significantly better than that of the model control group. In contrast, the positive drug hydroxychloroquine did not show any trend of saliva improvement after 2 weeks of administration, and only after 3 weeks of administration could it show a statistical difference compared with the model group, indicating that the time required for CP25M2 to take effect is shorter than that of the positive drug hydroxychloroquine. In addition, after 2 weeks of CP25M2 administration, the saliva secretion of each dose group increased significantly, while after 2 weeks of CP-25 administration, only the high-dose group showed an improvement in saliva secretion, indicating that CP25M2 acts quickly and can achieve the effect of improving saliva secretion more quickly.

[0112] Sjögren's syndrome is a lingering disease, and patients are troubled by strong dryness symptoms for a long time. The oral mucosa, teeth, lips and tongue are sticky due to lack of saliva. They need to drink water frequently when speaking. In severe cases, they cannot eat solid food without water. The quality of daily life is greatly affected, so there is an obvious demand for the onset of the drug. In addition, due to the relatively long course of Sjögren's syndrome, the medication time may be relatively long. For safety reasons, low-dose administration is a relatively suitable medication regimen. It can be seen that patients with Sjögren's syndrome have a demand for medication that is fast-acting and low-dose. Example 8 shows that CP25M2 can show significant efficacy after 2 weeks of low-dose administration, and the onset of male model animals is better than CP-25 and positive control drugs, suggesting that CP25M2 is more in line with the ideal medication expectations of fast onset and low dosage for patients with Sjögren's syndrome.

[0113] In general, CP25M2 has high solubility and fast dissolution rate, and its formulation feasibility is significantly better than CP-25. It also has a definite therapeutic effect on Sjögren's syndrome. Low-dose administration can quickly show an effect of improving saliva secretion, and its onset speed is better than that of the positive drug hydroxychloroquine, showing good potential for the development of Sjögren's syndrome drugs.

[0114] The above disclosure is only the preferred embodiment of the present invention, which cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. Debenzoylpeonidin-6'-O-benzenesulfonate, characterized in that: It has the following chemical structure:

2. A method for preparing debenzoylpaeoniflorin-6'-O-benzenesulfonate according to claim 1, characterized in that: The steps include: A. dissolving paeoniflorin-6'-O-benzenesulfonate in an alkaline solvent and stirring for reaction; B. After the reaction is completed, the system is adjusted to neutral or acidic, and the solvent is removed to obtain crude product-Ⅰ; C. Dissolving the crude product I in water, extracting with an organic solvent, and taking the aqueous phase; D. Concentrating the aqueous phase, performing column chromatography or preparative liquid separation, collecting the eluate, and removing the solvent to obtain the debenzoylpaeoniflorin-6'-O-benzenesulfonate.

3. The method for preparing debenzoylpaeoniflorin-6'-O-benzenesulfonate according to claim 2, characterized in that: In step A, the alkaline solvent is 1 to 100 times the weight of paeoniflorin-6'-O-benzenesulfonate, the pH of the system is adjusted to 10 to 12, the stirring reaction temperature is room temperature to 60°C, the reaction environment is -0.1 MPa to normal pressure, and the reaction time is 15 min to 48 h.

4. The method for preparing debenzoylpaeoniflorin-6'-O-benzenesulfonate according to claim 3, characterized in that: In step A, the weight of the alkaline solvent is 50 times that of paeoniflorin-6'-O-benzenesulfonate, the stirring reaction temperature is room temperature, the reaction environment is normal pressure, and the reaction time is 48 hours.

5. The method for preparing debenzoylpaeoniflorin-6'-O-benzenesulfonate according to claim 3, characterized in that: In step A, the alkaline solvent is 50 times the weight of paeoniflorin-6'-O-benzenesulfonate, the stirring reaction temperature is 45° C., the reaction environment pressure is not higher than -0.08 MPa during the reaction, and the reaction time is 2 h.

6. The method for preparing debenzoylpaeoniflorin-6'-O-benzenesulfonate according to claim 2, characterized in that: In step A, the alkali source in the alkaline solvent includes one or more of sodium hydroxide, potassium hydroxide, sodium methoxide, potassium carbonate, diethylamine, triethylamine, and ammonia water, and the solvent includes one or more of methanol, ethanol, isopropanol, acetone, ethyl acetate, and water; in step B, one or more of hydrochloric acid and sulfuric acid are added dropwise to adjust the system to neutrality, and the method for removing the solvent includes reduced pressure concentration, freeze drying, and spray drying; in step C, the organic solvent includes one or more of ether, petroleum ether, ethyl acetate, chloroform, and dichloromethane; in step D, the method for concentrating the aqueous phase includes reduced pressure concentration, freeze drying, and spray drying; the filler used for column chromatography or preparative liquid phase separation includes one of silica gel and macroporous resin, the eluent used includes one or more of methanol, ethanol, isopropanol, acetonitrile, water, chloroform, and dichloromethane, and the method for removing the solvent includes reduced pressure concentration, freeze drying, and spray drying.

7. The method for preparing debenzoylpaeoniflorin-6'-O-benzenesulfonate according to claim 6, characterized in that: In step A, in the alkaline solvent, the alkali source is sodium hydroxide, and the solvent is methanol or water; in step A, when the alkaline solvent contains water, paeoniflorin-6'-O-benzenesulfonate is first dissolved in a non-aqueous solvent and then mixed with the alkaline solvent; in step B, 0.1-1M hydrochloric acid is added dropwise to adjust the system to neutral or acidic; in step C, the organic solvent is ether or chloroform; in step D, the aqueous phase is first adjusted to neutral.

8. The method for preparing debenzoylpaeoniflorin-6'-O-benzenesulfonate according to claim 6, characterized in that: In step D, the filler used for column chromatography or preparative liquid separation is silica gel, and the eluent used is chloroform:methanol=15-10:

1.

9. The method for preparing debenzoylpaeoniflorin-6'-O-benzenesulfonate according to claim 6, characterized in that: In step D, the filler used for column chromatography or preparative liquid separation is ODS or macroporous resin, and the eluent used is 10% to 30% acetonitrile-water solution.

10. A use of debenzoylpaeoniflorin-6'-O-benzenesulfonate as claimed in claim 6, characterized in that: Application in the preparation of medicines for treating autoimmune diseases; the autoimmune disease is Sjögren's syndrome.

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