CN106977561B

CN106977561B - Preparation of Sutherlandin-5-p-hydroxybenzoate and application thereof in preparation of drugs for treating rheumatoid arthritis

Info

Publication number: CN106977561B
Application number: CN201610028681.1A
Authority: CN
Inventors: 曲桂武; 刘俊杰; 吴长景; 曹奇志; 孙亚楠; 伊鹏; 于泽秋
Original assignee: Binzhou Medical College
Current assignee: Binzhou Medical College
Priority date: 2016-01-16
Filing date: 2016-01-16
Publication date: 2021-04-20
Anticipated expiration: 2036-01-16
Also published as: CN106977561A

Abstract

The invention relates to a chemical structure of a compound Sutherlandin-5-p-hydroxybenzoate, a preparation method and application of the compound in preparing a medicament for treating rheumatoid arthritis. Sutherlandin-5-p-hydroxybenazolate is a novel compound purified and prepared from traditional Chinese medicine, namely, pearl plum Sorbaria sorbifolia (L.) A.Brown, by the applicant of the invention, and can obviously inhibit PEG in serum of adjuvant arthritis rats₂The tripterygium glycosides with the level of NO and the activity higher than that of tripterygium glycosides with the same dosage can be used for preparing the medicine for treating the rheumatoid arthritis. The chemical structure, the preparation method and the anti-RA activity of the compound are disclosed for the first time, so that the compound has prominent substantive characteristics.

Description

Preparation of Sutherlandin-5-p-hydroxybenzoate and application thereof in preparation of drugs for treating rheumatoid arthritis

Technical Field

The invention relates to a structure and a preparation method of Sutherlandin-5-p-hydroxybenzoate, and application of Sutherlandin-5-p-hydroxybenzoate in preparing a medicament for treating rheumatoid arthritis.

Background

Rheumatoid Arthritis (RA) is a heterogeneous, systemic, autoimmune disease with symmetric polyarthritis as the main clinical manifestation, and is a highly heterogeneous autoimmune disease under the interaction of environmental factors, genetic background, and both. RA is one of the main reasons for the loss of labor capacity and disability of human beings, the average incidence rate of the diseases is 0.2-1.2% all over the world, and the prevalence rate of China is 0.2-0.93%. The treatment of RA patients requires long-term administration to control the course of disease and improve symptoms, and has high treatment cost and heavy social burden. At present, the drugs clinically used for treating RA mainly comprise non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, disease-modifying antirheumatic drugs, biological agents, botanical drug preparations and the like, and although the drugs have certain curative effects, the drugs have strong toxic and side effects, which has a great difference from the ideal antirheumatic drugs which need to be taken for a long time. Therefore, the compound with the anti-RA activity which is safer and more efficient has great value in the aspect of developing RA treatment medicines.

The compound Sutherlandin-5-p-hydroxybenzoate is a new compound purified and prepared from traditional Chinese medicine, namely, pearl plum Sorbaria sorbifolia (L.) A.Brown. The chemical structure, the preparation method and the anti-RA activity of the compound are disclosed for the first time, so that the compound has prominent substantive characteristics.

Disclosure of Invention

The invention provides a preparation method of a novel compound and application of the novel compound in preparing a medicament for treating rheumatoid arthritis.

The invention relates to a compound for preparing rheumatoid arthritis, which is Sutherlandin-5-p-hydroxybenzoate and has the chemical formula: c₁₈H₂₁NO₉The structural formula is:

the preparation method of Sutherlandin-5-p-hydroxybenzoate related by the invention comprises the following steps:

taking a medicinal material pearl plum, crushing into coarse powder, adding 20 times of organic alcohol aqueous solution with proper concentration as an extraction solvent, soaking and extracting for 1-3 times, each time being about 10 hours, combining extracting solutions, filtering, dealcoholizing and concentrating until the concentration of the organic alcohol is about 20%, loading the mixture on a macroporous adsorption resin column, eluting 5 column volumes by using about 20% organic alcohol aqueous solution to remove impurities, eluting 4 column volumes by using 50-95% organic alcohol, collecting 50-95% organic alcohol eluting parts, drying under reduced pressure, loading the mixture on a 100-200-mesh silica gel column, selecting a proper silica gel chromatography eluting system to elute, collecting an eluting part containing Sutherlandin-5-p-hydroybenzoate, concentrating, crystallizing and filtering to obtain Sutherlandin-5-p-hydroybenzoate coarse crystals. The crude crystal is dissolved in a recrystallization solvent to form a supersaturated solution, and the solution is recrystallized to obtain a Sutherlandin-5-p-hydroxybenzoate sample with the purity of more than 98%.

The organic alcohol used as the extraction solvent in the process is one of methanol and ethanol, preferably ethanol.

The macroporous adsorption resin column involved in the process is low-polarity or non-polar macroporous adsorption resin, and low polarity is preferred.

The silica gel chromatography elution system related in the process is one or more of dichloromethane, ethyl acetate, n-butanol, methanol and petroleum ether, and a dichloromethane-methanol system is preferred.

The recrystallization solvent involved in the process can be one or more of methanol, ethanol, acetone, ethyl acetate, chloroform and the like, and preferably methanol or ethyl acetate.

The invention also provides application of the compound in preparing a medicament for treating rheumatoid arthritis.

The chemical structure, the preparation method and the anti-rheumatoid arthritis activity of the compound are disclosed for the first time, the possibility of any inspiration given by other compounds does not exist, the compound has prominent substantive characteristics, and the compound is expected to be used for developing a novel anti-rheumatoid arthritis treatment drug.

Drawings

FIG. 1 is a Sutherlandin-5-p-hydroxybenzoate positive ion mass spectrum (HR-ESI-MS).

FIG. 2 is a UV spectrum of Sutherlandin-5-p-hydroxybenzoate (solvent: CD)₃OD)。

FIG. 3 is Sutherlandin-5-p-hydroxybenzoate¹H NMR Spectrum (solvent: CD)₃OD)。

FIG. 4 is Sutherlandin-5-p-hydroxybenzoate¹³C NMR Spectroscopy (solvent: CD)₃OD)。

FIG. 5 is a Sutherlandin-5-p-hydroxybenzoate DEPT spectrum (solvent: CD)₃OD)。

FIG. 6 is Sutherlandin-5-p-hydroxybenzoate¹H-¹H COSY spectrum (solvent: CD)₃OD)。

FIG. 7 shows the Sutherlandin-5-p-hydroxybenzoate HMQC spectrum (solvent: CD)₃OD)。

FIG. 8 is Sutherlandin-5-p-hydroxybenZoate HMBC Spectroscopy (solvent: CD)₃OD)。

FIG. 9 is a Sutherlandin-5-p-hydroxybenzoate NOESY spectrum (solvent: CD)₃OD)。

Detailed Description

The following examples are intended to illustrate the invention, but not to limit the substance of the invention.

Example 1 preparation of Sutherlandin-5-p-hydroxybenzoate

Taking a medicinal material of pearl plums, crushing into coarse powder, adding 20 times of 95% ethanol, soaking and extracting for 1-3 times, each time for about 10 hours, combining extracting solutions, filtering, dealcoholizing and concentrating until the ethanol concentration is about 20%, loading the mixture on an AB-8 type weak-polarity macroporous adsorption resin column, eluting 5 column volumes by using 20% ethanol solution to remove impurities, eluting 4 column volumes by using 95% ethanol, collecting 95% ethanol elution parts, drying under reduced pressure, then loading the mixture on a 100-200-mesh silica gel column, taking dichloromethane-methanol as an elution system, carrying out stage elution according to the methanol concentration from low to high, collecting the elution parts with the methanol concentration of 20-30%, concentrating, crystallizing, filtering, drying under reduced pressure, and obtaining sutherland-5-p-hydroybenzoate coarse crystals. And dissolving the coarse crystals in ethyl acetate to form a supersaturated solution, recrystallizing, filtering, and drying under reduced pressure to obtain the product.

Example 2 preparation of Sutherlandin-5-p-hydroxybenzoate

Taking a medicinal material of pearl plums, crushing into coarse powder, adding 20 times of methanol, soaking and extracting for 1-3 times, extracting for about 10 hours each time, combining extracting solutions, filtering, dealcoholizing and concentrating to a proper volume, replenishing water to about 20% of alcohol concentration, loading on a D-101 type nonpolar macroporous adsorption resin column, eluting 5 column volumes by using 20% methanol solution to remove impurities, eluting 4 column volumes by using 90% methanol, collecting 90% methanol elution parts, drying under reduced pressure, loading on a 100-200-mesh silica gel column, taking dichloromethane-methanol as an elution system, carrying out stage elution according to the methanol concentration from low to high, collecting the elution parts with the methanol concentration of 20-30%, concentrating, crystallizing, filtering, and drying under reduced pressure to obtain sutherland-5-p-hydroxybenzoate coarse crystals. And dissolving the coarse crystals in ethyl acetate to form a supersaturated solution, recrystallizing, filtering, and drying under reduced pressure to obtain the product.

Example 3 structural confirmation of Sutherlandin-5-p-hydroxybenzoate

1. Apparatus and materials

Jasco P-1020 digital polarimeter, Agilent TOF/6500 high resolution Mass Spectrometry, Shimadzu UV-2401 visible-ultraviolet Spectrophotometer, Bruke Avance III 500NMR spectrometer, melting point determinator Yanaco MP53 model (melting point uncorrected). Sutherlandin-5-p-hydroxybenzoate samples were prepared as described above in example 1.

2. Structure identification of compounds

Colorless needle crystal (methanol), mp 79-81 deg.C, is easily soluble in dimethyl sulfoxide and methanol, is slightly soluble in ethyl acetate, acetone, chloroform, water, and is insoluble in petroleum ether,

positive ion ESI-MS m/z: 396[ M + H]⁺，418[M+Na]⁺(ii) a Negative ion ESI-MS m/z: 394[ M-H]^-，430[M+Cl]^-，789[2M-H]^-. Positive ion HR-ESI-MS m/z: found 418.1104[ M + Na]⁺Calculated value 418.1114 (C)₁₈H₂₁NO₉Na[M+Na]⁺) (ii) a Negative ion HR-ESI-MS m/z: found 394.1139[ M-H ]]^-Calculated value 394.1138 (C)₁₈H₂₀NO₉[M-H]^-)。UVλ_maxnm(logε)in MeOH：259(3.29)，210(3.49)，198(3.52)。¹H-NMR(500Hz,CD₃OD)δ：7.93(2H,br d,J＝8.8Hz,H-3',7')，6.85(2H,br d,J＝8.8Hz,H-4',6')，5.74(1H,br s,H-2)，5.09(2H,AB type,Ha-5)，4.73(1H,br d,J＝13.6Hz,Ha-4)，4.64(1H,br d,J＝13.6Hz,Hb-4)，4.35(1H,d,J＝7.8Hz,H-1”)，3.88(1H,dd,J＝12.0,2.2Hz,Ha-6”)，3.70(1H,dd,J＝12.0,5.2Hz,Hb-6”)，3.38(1H,t,J＝8.6Hz,H-3”)，3.34(1H,t,J＝8.6Hz,H-4”)，3.30(1H,ddd,J＝9.8,5.2,2.2Hz,H-5”)，3.24(1H,dd,J＝8.6,7.8Hz,H-2”)。¹³C-NMR(125Hz,CD₃OD)δ：167.1(s,C-1')，164.0(s,C-5')，159.8(s,C-3)，133.1(d,C-3',7')，121.4(s,C-2')，116.5(s,C-1)，116.4(d,C-4',6')，104.3(d,C-1”)，97.7(d,C-2)，78.1(d,C-5 "), 78.0(d, C-3"), 74.9(d, C-2 "), 71.4(d, C-4"), 68.4(t, C-4), 64.6(t, C-5), 62.5(t, C-6 "). The NMR data are based on DEPT spectra and¹H-¹and attributing the two-dimensional spectrum data analysis results of H COSY, HMQC, HMBC and the like. It is concluded from this that the compound structure is:

example 4 preparation of Sutherlandin-5-p-hydroxybenzoate tablets

Taking 10 g of Sutherlandin-5-p-hydroybenzoate, adding a proper amount of 95% ethanol to form an extract, then adding cyclodextrin or porous starch, uniformly mixing to form a soft material, granulating by using a granulator, carrying out vacuum drying, carrying out size stabilization, then adding a lubricant and a flavoring agent, mixing and tabletting, wherein the tablet weight is 250-500 mg, and each tablet contains 5-10 mg of Sutherlandin-5-p-hydroybenzoate.

Example 5 preparation of Sutherlandin-5-p-hydroxybenzoate capsules

Taking 10 g of Sutherlandin-5-p-hydroxybenzoate, adding starch and sodium carboxymethylcellulose, uniformly mixing, and filling into capsules to obtain the compound. Each capsule contains 5-10 mg of Sutherlandin-5-p-hydroxybenzoate.

Example 6 Sutherlandin-5-p-Hydroxybenzoate adjuvant treatment of PEG in serum of arthritis rats₂Effect on NO level

1. Apparatus and materials

Ex1800 model enzyme linked immunoassay analyzer (Biotek); UV-2450 type spectrophotometer (Shimadzu, Japan). Bcg, shanghai biologies institute; lanolin, paraffin oil, NO kit and prostaglandin E₂(PGE₂) The kit is purchased from Nanjing construction reagent company; tripterygium glycosides tablets, Shanghai Compound DAN Fuhua medicinal products, lot number 141001. Wistar rats, SPF grade, female, body mass 120-150 g, provided by Shandong green leaf pharmaceutical laboratory animal center.

2. Modeling and drug delivery

Rats were randomly divided into 4 groups (8 per group) of control, positive control Tripterygium glycosides, model and Sutherlandin-5-p-hydroybenzoate groups. The control group rats were injected with normal saline intradermally into the right hind foot sole, and other groups were injected with 0.1mL of Freund's complete adjuvant (each 10mg of BCG was dissolved in 1mL of paraffin oil, mixed well and autoclaved) intradermally into the right hind foot sole, and the circumference of the left hind foot ankle joint of the rats was recorded. The ig administration of each administration group is started on the 9 th day after the model building, the cyanogen glycoside group and the tripterygium glycosides are both administered according to 10mg/kg, the same amount of normal saline is administered to the model group and the control group, and the administration of each group is continuously performed for 13 d.

3. Index detection

Collecting blood from heart after last administration (21 days after molding) lh, collecting serum, storing at-20 deg.C, and measuring NO and PGE in serum according to kit instructions₂The amount of (c). The results are shown in Table 1.

TABLE 1 Sutherlandin-5-p-hydroxybenzoate adjuvant in rats' serum PEG for arthritis₂Influence of NO level (

n＝8)

4. And (4) conclusion: sutherlandin-5-p-hydroxybenzoate can obviously inhibit PEG in serum of adjuvant arthritis rat₂The tripterygium glycosides with the level of NO and the activity higher than that of tripterygium glycosides with the same dosage can be used for preparing the medicine for treating the rheumatoid arthritis.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Claims

1. A novel compound, Sutherlandin-5-p-hydroxybenzoate, has the following structural formula:

2. a process for the preparation of the compound of claim 1, characterized by:

pulverizing the medicinal material pearl plum into coarse powder, adding 20 times of organic alcohol water solution with proper concentration as extraction solvent, soaking and extracting for 1-3 times, each time for 10 hours, combining the extracting solutions, filtering, dealcoholizing, concentrating to 20% of organic alcohol concentration, loading to a macroporous adsorption resin column, eluting 5 column volumes by using a 20% organic alcohol aqueous solution to remove impurities, eluting 4 column volumes by using 50-95% organic alcohol, collecting 50-95% organic alcohol elution parts, drying under reduced pressure, loading to a 100-200 mesh silica gel column, selecting a proper silica gel chromatography elution system for elution, collecting an elution part containing sutherland-5-p-hydroxbenzoate, concentrating, crystallizing, filtering, obtaining the crude crystal of Sutherlandin-5-p-hydroxybenzoate, dissolving the crude crystal in a recrystallization solvent to form a supersaturated solution, recrystallizing, the Sutherlandin-5-p-hydroxybenzoate sample with the purity of more than 98 percent can be obtained.

3. The production method according to claim 2, characterized in that: the organic alcohol used as the extraction solvent is one of methanol and ethanol.

4. The production method according to claim 3, characterized in that: the organic alcohol used as the extraction solvent is ethanol.

5. The production method according to claim 2, characterized in that: the macroporous adsorption resin column involved in the process is low-polarity or non-polar macroporous adsorption resin.

6. The production method according to claim 5, characterized in that: the macroporous adsorption resin column involved in the process is weak-polarity macroporous adsorption resin.

7. The production method according to claim 2, characterized in that: the silica gel chromatography elution system related by the process is one or more of dichloromethane, ethyl acetate, n-butanol, methanol and petroleum ether.

8. The production method according to claim 7, characterized in that: the silica gel chromatography elution system related to the process is a dichloromethane-methanol system.

9. The production method according to claim 2, characterized in that: the recrystallization solvent involved in the process can be one or more of methanol, ethanol, acetone, ethyl acetate and chloroform.

10. The production method according to claim 9, characterized in that: the recrystallization solvent involved in the process is methanol or ethyl acetate.

11. Use of a compound according to claim 1 for the manufacture of a medicament for the treatment of rheumatoid arthritis.