Arbutinone acid derivatives with methoxy substitution, preparation method and application

Abstract

The present invention belongs to the field of pharmaceutical technology, and discloses a bearberry ketonic acid derivative with a methoxy substitution, a preparation method and an application. By using a microbial method, the present invention uses bearberry ketonic acid as a substrate, specifically introduces a methoxy group at the 11th position of the bearberry ketonic acid nucleus, and simultaneously introduces a hydroxyl group at the 21β position to obtain 3-carbonyl-21-hydroxy-11-methoxy-12-ene-ursane-28-carboxylic acid. Specifically, the carbonyl group at the 3rd position of bearberry ketonic acid is cleaved to form a methoxy group, and at the same time, a double bond at the 2(3) position is formed to form 3-methoxy-2(3),12(13)-diene-ursane-28-carboxylic acid. It is confirmed by in vitro anti-inflammatory experiments that the compounds of formula I and formula II have good anti-inflammatory activities, can be used as active ingredients of anti-inflammatory drugs, and have a wide range of uses.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a bearberry ketone acid derivative with a methoxy substitution, a preparation method and an application thereof. Background Art

[0002] Inflammation is a complex defense reaction of the body against harmful stimuli. However, excessive inflammatory responses can cause damage to the body and are associated with various diseases. Currently, the commonly used anti-inflammatory drugs in clinical practice are mainly divided into steroid anti-inflammatory drugs and non-steroidal anti-inflammatory drugs. Steroid anti-inflammatory drugs are hydrocortisone, a glucocorticoid secreted by the adrenal cortex, and its synthetic derivatives. However, long-term use of glucocorticoids can cause a variety of serious adverse reactions. Therefore, steroid anti-inflammatory drugs are usually not used for the conventional treatment of mild inflammatory responses and are mainly used for the treatment of severe inflammatory response diseases. Non-steroidal anti-inflammatory drugs, that is, the widely used antipyretic, analgesic and anti-inflammatory drugs in clinical practice, are mainly used for the treatment of fever, pain and rheumatoid arthritis, and have the effects of rapidly reducing inflammatory responses, relieving pain and improving the body's functions. However, they are usually not suitable for the preventive treatment of diseases and complications. Long-term use of such drugs can cause side effects such as gastrointestinal discomfort and kidney damage, and gastrointestinal mucosal damage is the most common. In recent years, natural drugs have attracted the attention of domestic and foreign researchers due to their multi-target effects. It has been found that the active ingredients of a variety of natural drugs show varying degrees of anti-inflammatory effects through their mechanisms of action. Among them, the anti-inflammatory activity of triterpenoids has attracted the attention of domestic and foreign researchers due to their multi-target effects. Triterpenes and their glycosides are an important class of natural organic compounds widely present in nature. It is reported that most of these compounds have good anti-inflammatory activity. Such compounds have good inhibitory effects on acute and chronic inflammation, can play an anti-inflammatory role by inhibiting the production of inflammatory factors, and in addition, the anti-inflammatory effect of such compounds is also related to their ability to affect the immune system.

[0003] However, due to the complex molecular structure of triterpenes, problems such as poor hydrophilicity and low bioavailability of compounds severely limit their effective use as drugs in clinical practice. Currently, there are mainly two common methods to improve the bioavailability of triterpenoid compounds. One is to use chemical synthesis methods to modify and transform their structures to improve the bioactivity and hydrophilicity of the compounds. However, due to the special structure of pentacyclic triterpenoid compounds, their parent nucleus itself lacks reactive groups for chemical reactions. Therefore, it severely limits the reaction sites for chemical structure modification of triterpenoid compounds and the diversity of derivative preparation. Another method is biotransformation, which utilizes the catalytic activity of microorganisms with mild reaction conditions and high regioselectivity and stereoselectivity. Thus, it has become a powerful tool for the structure modification and transformation of complex natural products. On the one hand, biotransformation products can obtain derivatives with stronger biological activity directly for drug research and development. On the other hand, the newly introduced chemical reactive groups on the parent nucleus after microbial transformation increase the sites for chemical modification and transformation, solving the problem of few reaction sites for the preparation of derivatives of triterpenoid compounds by organic chemistry. However, the method of microbial transformation has strong randomness and cannot design and predict products like chemical synthesis methods. Therefore, screening suitable microbial strains plays a decisive role in the structure of the transformation products. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a methoxy-substituted ursolic acid derivative or a pharmaceutically acceptable salt thereof and a preparation method thereof, and the ursolic acid derivative can be used to prepare anti-inflammatory drugs.

[0005] The methoxy-substituted ursolic acid derivative provided by the present invention is a compound with the structural formula of formula I, 3-oxo-21β-hydroxy-11α-methoxy-12-ene-ursane-28-carboxylic acid and a compound with the structural formula of formula II, 3-methoxy-2(3),12(13)-dien-ursane-28-carboxylic acid:

[0006]

[0007] The compounds with the structural formulas of formula I and formula II are ursolic acid derivatives first disclosed by the present invention.

[0008] The present invention also provides a preparation method of the above-mentioned ursolic acid derivative, including the following steps:

[0009] 1) Ferment and culture microorganisms, add ursolic acid to the culture medium, then carry out transformation culture, and obtain the fermentation broth after removing the mycelium. The microorganisms are strains of the genus Aspergillus;

[0010] 2) After extracting the fermentation broth, evaporate the extract to dryness to obtain a crude transformation extract;

[0011] 3) The crude extract was subjected to silica gel column chromatography, and gradient elution was carried out using dichloromethane: methanol as the mobile phase. After collecting the fractions, 5 components were obtained by HPLC analysis and combined.

[0012] 4) The said components were purified by reverse-phase high-performance liquid chromatography to obtain the compounds with the structural formulas of Formula I and Formula II.

[0013] The present invention also provides the use of the above arbutinone acid derivative or the arbutinone acid derivative prepared by the above preparation method in the preparation of anti-inflammatory drugs.

[0014] Compared with the prior art, the present invention utilizes the microbial transformation technology to specifically introduce a methoxy group at the 11α position of the arbutinone acid nucleus and simultaneously introduce a hydroxyl group at the 21β position to form a transformation product with the structural formula of Formula I. Specifically, the carbonyl group at the 3 position of arbutinone acid is cleaved to form a methoxy group, and at the same time, a double bond at the 2(3) position is formed to form a transformation product with the structural formula of Formula II. Through in vitro anti-inflammatory experiments, it is confirmed that Compounds I and II have good anti-inflammatory activities and can be used as active ingredients of anti-inflammatory drugs, with wide applications. Detailed Embodiments

[0015] To further illustrate the present invention, the arbutinone acid derivative with methoxy substitution and its preparation method provided by the present invention will be described in detail below in conjunction with the embodiments.

[0016] Example 1: Preparation of Compounds with the Structural Formulas of Formula I and Formula II

[0017] The present invention adopts the microbial transformation method. Using arbutinone acid as the raw material, the compounds of the present invention are prepared through steps such as fermentation, extraction, and separation. Strains of the genus Aspergillus can be purchased from the China General Microbiological Culture Collection Center (CGMCC), and potato medium is selected and stored in a 4°C refrigerator on a solid slant medium.

[0018] Taking Aspergillus oryzae CGMCC 3.2073 as an example, the process for preparing the compounds with the structural formulas of Formula I and Formula II is as follows:

[0019] 1) Fermentation, Transformation, and Extraction

[0020] Aspergillus oryzae CGMCC 3.2073 was inoculated into two 250 mL Erlenmeyer flasks (containing 100 mL of potato medium) as the seed solution. After shaking culture at 160 rpm and 26 °C for 12 hours, when the mycelial growth was in the vigorous stage, 1 mL of the seed solution was pipetted with a sterile pipette and added to twenty 1000 mL shake flasks (containing 400 mL of potato medium). After shaking culture for 24 hours, 20 mg of arbutinone acid (0.2 mL, 100 mg / mL ethanol solution) was added to each shake flask, and a total of 400 mg of the substrate was used. The transformation was continued for 3 days under the same conditions. The fermentation broth was filtered to remove the mycelium, and the filtrate was extracted 3 times with an equal volume of ethyl acetate. The extract was concentrated under reduced pressure to dryness to obtain approximately 0.85 g of the crude transformation product.

[0021] 2) Silica gel column chromatography separation

[0022] The crude transformation product was separated by silica gel column chromatography. Dichloromethane: methanol gradient elution (100:1, 50:1, 20:1, 5:1). The fractions were collected, combined after HPLC analysis, and combined fractions A and B were obtained.

[0023] 3) Reverse-phase high-performance liquid chromatography purification

[0024] Fraction A was purified by reverse-phase high-performance liquid chromatography. The preparation conditions were a semi-preparative chromatographic column YMC ODS A-5μm, 10.0×250 mm, methanol / water (92:8, 3.0 mL / min), detection wavelength 203 nm, and the transformed product with the structure shown in Formula I was obtained. Fraction B was purified by reverse-phase high-performance liquid chromatography, using the mobile phase methanol / water (73:27, 3.0 mL / min), detection wavelength 203 nm, and the transformed product with the structure shown in Formula II was obtained. The mass spectrometry and spectroscopy data of Compounds I and II are shown below.

[0025] Compound I: 3-oxo-21β-hydroxy-11α-methoxy-12-ene-ursane-28-carboxylic acid; main absorption peaks in the infrared spectrum (KBr): ν max 3594, 3048, 2981, 1761, 1713, 1386, 1235, 1052 cm -1 ; high-resolution mass spectrometry [M-H] - m / z was 499.3433 (cal. 499.3423, C 31 H 47 O5); the nuclear magnetic resonance hydrogen spectrum and carbon spectrum data are shown in Table 1.

[0026] Compound II: 3-methoxy-2(3),12(13)-dien-ursane-28-carboxylic acid; main absorption peaks in the infrared spectrum (KBr): ν max3673, 3027, 2952, 1715, 1386, 1257, 1063 cm -1 ; High-resolution mass spectrometry m / z [M-H] - m / z is 467.3534 (cal. 467.3525, C 31 H 47 O3); The 1H NMR and 13C NMR data are shown in Table 1.

[0027] Table 1. 1H NMR and 13C NMR data of Compounds I and II

[0028]

[0029]

[0030] The above results indicate that the structures of the obtained compounds of Formula I - Formula VII are correct.

[0031] Example 2 Anti-inflammatory activities of Compound I and Compound II of the present invention

[0032] 1) Experimental materials

[0033] Instruments and reagents: CO2 incubator (Jouan IGO150); microplate reader (Bio-TEK ELx800); fluorescence inverted microscope (Olympus IX51); MTT cell proliferation and cytotoxicity detection kit (Beyotime Biotechnology Research Institute), RPMI1640 medium (Gibcol BRL), mouse mononuclear macrophages RAW264.7, Rnase A, fetal bovine serum, dimethyl sulfoxide (DMSO), trypsin (Shanghai Bioengineering Co., Ltd.).

[0034] Test samples: Ursolic acid and Compound I and Compound II obtained in Example 1, with a purity of more than 95%; at the same time, quercetin was selected as the positive control drug, and each compound was dissolved in DMSO and diluted.

[0035] 2) Experimental method

[0036] The MTT method was used to determine the effects of the test compounds on the cell viability of mouse mononuclear macrophages RAW264.7: RAW264.7 cells in the logarithmic growth phase were taken, and the cell concentration was adjusted to 5×10 4Cells were inoculated at a density of [number] / mL into a 96-well culture plate. In the drug treatment group and the cell control group, 100 μL of cell suspension was added to each well, with 3 replicates in each group. The blank control group was only added with 100 μL of DMEM complete medium per well, with 3 replicates. After culturing the 96-well culture plate in an incubator at 37 °C and 5% CO2 for 24 h, different concentrations of the test samples were added to make the final concentration 0.1 - 100 μM, and the culture was continued for 72 h. According to the MTT method, the absorbance (A) value at 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the inhibition rate was calculated [Inhibition rate = (1 - A value of the experimental group / A value of the control group) × 100%].

[0037] The Griess method was used to determine the effect of each test compound on the release of NO from LPS-induced RAW264.7 cells: The cell concentration was adjusted to 2×10 5 / mL and inoculated into a 96-well culture plate, with 1 mL of cell suspension in each well and 3 replicates in each group. The blank control group was only added with DMEM complete medium, with 3 replicates. After culturing the 96-well culture plate in an incubator at 37 °C and 5% CO2 for 24 h, different concentrations of the test samples were added to make the final concentration 0.1 - 100 μM. After continuing the culture for 12 h, the supernatant was taken and the level of NO in the culture medium was measured according to the instructions of the kit. The data was analyzed and processed using SPSS Statistics 25 software.

[0038] 3) Experimental results

[0039] According to the test results of the MTT method and the Griess method, the effects of ursolic acid, Compound I and Compound II of the present invention on the release of NO from LPS-induced RAW264.7 cells were calculated, and the results are shown in Table 2.

[0040] Table 2. Results of the test samples inhibiting the release of NO from LPS-induced RAW264.7 cells

[0041]

[0042] The results showed that neither Compound I nor Compound II of the present invention showed obvious cytotoxic activity against RAW264.7 cells. At the same time, they could significantly reduce the release level of the inflammatory factor NO from LPS-induced RAW264.7 cells, and the inhibitory activity was significantly better than that of the positive control quercetin, having significant anti-inflammatory activity and could be used as the active ingredient of anti-inflammatory drugs.

[0043] Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A ursolic acid derivative having 21 β -hydroxy, 11 α -methoxy substitution, and the ursolic acid derivative is 3-oxo-21 β -hydroxy-11 α -methoxy-12-ene-ursane-28-carboxylic acid, having the structure of Formula I: 。 2. A ursolic acid derivative substituted with 3 - methoxy and 2(3) - double bond, wherein the ursolic acid derivative is 3 - methoxy - 2(3), 12(13) - di - double bond - ursane - 28 - carboxylic acid and has the structure of Formula II: 。 3. A method for preparing a bearberry quinone acid derivative as described in claim 1 or 2 by microbial transformation, characterized in that, including the following steps: 1) Ferment and culture microorganisms, add arbutin to the culture medium, then carry out transformation culture, and obtain the fermentation broth after removing the mycelium. The microorganisms are Aspergillus oryzae Aspergillus oryzae CGMCC 3.2073; 2) Extracting the fermentation broth to obtain a transformant; 3) Purifying the transformant by silica gel column chromatography, wherein the silica gel column purification uses gradient elution with a dichloromethane - methanol two - phase system, and collecting and combining the fractions; 4) Purify the said components by reverse-phase high performance liquid chromatography to obtain the ursolic acid derivatives, and the said ursolic acid derivatives are 3-oxo-21 β -hydroxy-11 α -methoxy-12-ene-ursane-28-carboxylic acid and 3-methoxy-2(3), 12(13)-diene-ursane-28-carboxylic acid.

4. Use of the ursolic acid derivative according to claim 1 or 2 or the ursolic acid derivative prepared by the preparation method according to claim 3 in the preparation of anti - inflammatory drugs.

Images