A bearberrylonic acid derivative, a method for preparing the same by microbial transformation and applications thereof

Abstract

The present invention belongs to the field of pharmaceutical technology, and discloses a ursolic acid derivative, a preparation method by microbial transformation and an application thereof. By using the microbial transformation technology, the present invention uses ursolic acid as a substrate, and regioselectively introduces a carbonyl group or a hydroxyl group at the C-21 position of the ursolic acid nucleus. The introduced hydroxyl group also has stereoselectivity, and is all β-hydroxyl. In addition, lactones can also be introduced at the 28th position and the 13β position, thereby obtaining novel ursolic acid derivatives of Formula I - Formula IV. It is confirmed by in vitro anti-tumor cell tests that the compounds of Formula I - Formula IV have significant anti-tumor activities, can be used as active ingredients of anti-tumor drugs, and have wide applications.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and relates to a bearberry ketonic acid derivative, a preparation method by microbial transformation and applications thereof, and particularly relates to a preparation method of a novel bearberry ketonic acid derivative with a carbonyl or hydroxyl substitution at the C-21 position and its application in the preparation of anti-tumor drugs. Background Art

[0002] Bearberry ketonic acid is a pentacyclic triterpenoid compound of the ursane type and is widely distributed in medicinal plants. Bearberry ketonic acid can also be extracted from common foods such as hawthorn, crabapple fruits and ripe jujube fruits. Among them, jujube is the main component of the traditional Chinese medicine formula PHY906 and is used for the adjuvant treatment of cancer patients. It has been found that bearberry ketonic acid is contained in plants such as the resin of Pistacia atlantica Desf. and Dipterocarpaceae trees, Trionyx sinensis, Piper nigrum and the roots of Ficus microcarpa. The wide presence of bearberry ketonic acid in medicinal plants indicates that bearberry ketonic acid itself has the potential to treat diseases such as cancer and infectious protozoa.

[0003] In terms of chemical structure, bearberry ketonic acid is the product of carbonylation at the C-3 position of ursolic acid and is an important intermediate for the chemical synthesis of ursolic acid derivatives. The research results show that derivatives with carbonylation at the C-3 position and cleavage of the A ring have various biological activities such as anti-tumor, anti-inflammatory, antibacterial, anti-protozoal, antioxidant, anti-diabetic and anti-viral. Due to the special structure of pentacyclic triterpenoid compounds, there are only two common chemical reaction active groups, namely the hydroxyl group at the C-3 position and the carboxyl group at the C-28 position, in the parent nucleus itself. At the same time, the existing research shows that the carbonyl group at the C-3 position is the active group for the anti-tumor activity of ursolic acid derivatives, which further limits the diversity of the preparation of ursolic acid derivatives with anti-tumor activity by using organic chemical synthesis methods.

[0004] Microbial transformation mainly uses the active enzymes produced by microbial systems as catalysts to modify specific structural sites of organic compound substrates in order to obtain new compounds with greater application value. Compared with traditional chemical synthesis methods, microbial transformation methods reduce complex reaction processes such as isomerization, racemization, rearrangement, and decomposition, with higher transformation efficiency, good chemoselectivity, good regioselectivity and stereoselectivity, milder reaction conditions, fewer by-products, simpler subsequent processing, and being more environmentally friendly. On the one hand, the transformation products obtained by microbial transformation can be used to obtain derivatives with stronger biological activity directly for drug research and development. On the other hand, the newly introduced chemical active groups on the parent nucleus after microbial transformation increase the sites for chemical modification and transformation, thus solving the problem of few reaction sites for the organic chemical preparation of derivatives of triterpenoid compounds. However, compared with chemical synthesis methods, the method of microbial transformation has great randomness. The transformation results cannot be predicted like those of chemical synthesis methods, nor can they be designed purposefully. At the same time, even similar substrates will have very significant differences in the products obtained after being catalytically transformed by the same strain or different strains. And the transformation products that can be obtained from the same substrate after being transformed by different strains may also have very significant differences. Therefore, it is crucial to select a suitable strain when using the method of microbial transformation for the structural modification of compounds. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a ursolic acid derivative having a carbonyl or hydroxyl substitution at the C-21 position or a pharmaceutically acceptable salt thereof and a method for preparing the same by microbial transformation. The ursolic acid derivative can be used to prepare anti-tumor drugs.

[0006] The present invention provides a ursolic acid derivative having a carbonyl or hydroxyl substitution at the C-21 position. The ursolic acid derivative is 3,21-dicarbonyl-12-ene-ursane-28-carboxylic acid, 3,21-dicarbonyl-7β-hydroxy-12-ene-ursane-28-carboxylic acid, 3-carbonyl-21β-hydroxy-11(12)-double bond-ursane-28,13β-lactone, and 3-carbonyl-7β,21β-dihydroxy-11(12)-double bond-ursane-28,13β-lactone, and the structures are shown in Formula I, Formula II, Formula III, and Formula IV respectively:

[0007]

[0008] The compounds with the structural formulas of Formula I - Formula IV are all novel ursolic acid derivatives first disclosed by the present invention.

[0009] The present invention also provides a method for preparing a ursolic acid derivative by microbial transformation, comprising the following steps:

[0010] 1) Ferment and culture the microorganism, add arbutinone acid to the culture medium, then carry out transformation culture, and obtain the fermentation broth after removing the mycelium. The microorganism is Aspergillus oryzae CGMCC 3.407;

[0011] 2) Extract the fermentation broth to obtain the transformant;

[0012] 3) Purify the transformant by reverse-phase silica gel column chromatography. The reverse-phase silica gel column purification uses a methanol-water two-phase system for gradient elution, and collect and combine the fractions;

[0013] 4) Purify the fractions by reverse-phase high-performance liquid chromatography to obtain 3,21-dioxo-12-ene-ursane-28-carboxylic acid, 3,21-dioxo-7β-hydroxy-12-ene-ursane-28-carboxylic acid, 3-oxo-21β-hydroxy-11(12)-double bond-ursane-28,13β-lactone, and 3-oxo-7β,21β-dihydroxy-11(12)-double bond-ursane-28,13β-lactone.

[0014] The synthetic route of the above microorganism transformation preparation method is shown in the following formula:

[0015]

[0016] The present invention also provides the application of the above arbutinone acid derivative or the arbutinone acid derivative prepared by the above preparation method in the preparation of anti-tumor drugs. The tumors include one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer, and colon cancer.

[0017] The present invention also provides the application of the above arbutinone acid derivative or the arbutinone acid derivative prepared by the above preparation method in the preparation of drugs for killing leukemia drug-resistant cells.

[0018] The present invention also provides an anti-tumor drug. The active ingredient of the anti-tumor drug is an arbutinone acid derivative or a pharmaceutically acceptable salt thereof; the arbutinone acid derivative is one or more of 3,21-dioxo-12-ene-ursane-28-carboxylic acid, 3,21-dioxo-7β-hydroxy-12-ene-ursane-28-carboxylic acid, 3-oxo-21β-hydroxy-11(12)-double bond-ursane-28,13β-lactone, and 3-oxo-7β,21β-dihydroxy-11(12)-double bond-ursane-28,13β-lactone; the tumors include one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer, and colon cancer.

[0019] Compared with the prior art, the present invention utilizes the catalytic action of microbial enzymes to regioselectively introduce a carbonyl group or a hydroxyl group at the 21st position of the arbutinone acid nucleus. The introduced hydroxyl group also has stereoselectivity, all being β-hydroxy groups. In addition, lactones can be introduced at the 28th position and the 13β position, thereby obtaining novel arbutinone acid derivatives of formula I - formula IV. It is confirmed by in vitro anti-tumor cell tests that the compounds of formula I - formula IV have significant anti-tumor activities and can be used as active ingredients of anti-tumor drugs, having a wide range of uses. Detailed implementation mode

[0020] To further illustrate the present invention, the preparation method of the novel arbutinone acid derivatives provided by the present invention and their applications in the preparation of anti-tumor drugs will be described in detail below in conjunction with examples.

[0021] Example 1: Preparation of compounds with the structural formula of formula I - formula IV

[0022] The present invention adopts a microbial transformation method, using arbutinone acid as a raw material, and preparing the compounds of the present invention 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.

[0023] Taking Aspergillus oryzae CGMCC 3.407 as an example, the process of preparing compounds with the structural formula of formula I - formula IV is as follows:

[0024] 1) Fermentation, transformation, and extraction

[0025] Aspergillus oryzae CGMCC 3.407 was inoculated into 2 250 mL Erlenmeyer flasks (containing 100 mL of potato medium) as seed solutions. 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 20 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 continued for 3 days under the same conditions. The fermentation broth was filtered to remove the mycelia, and the filtrate was extracted 3 times with an equal volume of ethyl acetate. The extract was concentrated under reduced pressure to dryness, and about 0.85 g of the crude transformation product was obtained.

[0026] 2) ODS-C18 column chromatography separation

[0027] The crude extract was separated by ODS-C18 column chromatography. Methanol: water gradient elution (10:90, 30:70, 60:40, 80:20, 100:0). The fractions were collected, combined after HPLC analysis, and the combined fractions Fr.1 - Fr.5 were obtained.

[0028] 3) Purification by reversed-phase high performance liquid chromatography

[0029] Fraction Fr.2 was purified by reversed-phase high performance liquid chromatography. The preparation conditions were a semi-preparative chromatographic column YMC ODSA - 5μm, 12.0×250mm, methanol - water (55:45, V / V), a flow rate of 3.0 mL / min, and a detection wavelength of 203 nm. The transformed products with the structural formulas of Formula I, Formula II, and Formula III were obtained. Fraction Fr.3 was purified by reversed-phase high performance liquid chromatography. The preparation conditions were a semi-preparative chromatographic column YMC ODSA - 5μm, 12.0×250mm, methanol - water (65:35, V / V), a flow rate of 3.0 mL / min, and a detection wavelength of 203 nm. The transformed product with the structural formula of Formula IV was obtained. The mass spectrometry and spectroscopy data of Compounds I - IV are shown below.

[0030] Compound I: 3,21-dioxo-12-ene-ursane-28-carboxylic acid; specific rotation (c = 0.1, MeOH); main absorption peaks of the infrared spectrum (KBr): ν max 3562, 2981, 1725, 1710, 1705, 1381, 1252, 1045 cm -1 ; high-resolution mass spectrometry [M - H] - m / z was 467.3174 (cal. 467.3161, C 30 H 43 O4); the 1H NMR and 13C NMR data are shown in Table 1.

[0031] Compound II: 3,21-dioxo-7β-hydroxy-12-ene-ursane-28-carboxylic acid; specific rotation (c = 0.1, MeOH); main absorption peaks of the infrared spectrum (KBr): ν max 3673, 3582, 2958, 1728, 1712, 1705, 1392, 1254, 1084 cm -1 ; high-resolution mass spectrometry [M - H] - m / z was 483.3126 (cal. 483.3110, C 30 H 43 O5); the 1H NMR and 13C NMR data are shown in Table 1.

[0032] Compound Ⅲ: 3-oxo-21β-hydroxy-11(12)-ene-ursane-28,13β-lactone; specific rotation (c = 0.1, MeOH); main absorption peaks of infrared spectrum (KBr): ν max 3596, 3043, 2967, 1725, 1711, 1699, 1348, 1293, 1097 cm -1 ; high-resolution mass spectrometry [M - H2O + H] + m / z is 451.3216 (cal. 451.3212, C 30 H 43 O3); 1H-NMR and 13C-NMR data are shown in Table 1.

[0033] Compound Ⅳ: 3-oxo-7β,21β-dihydroxy-11(12)-ene-ursane-28,13β-lactone; specific rotation (c = 0.1, MeOH); main absorption peaks of infrared spectrum (KBr): ν max 3667, 3511, 2969, 1727, 1715, 1325, 1258, 1066 cm -1 ; high-resolution mass spectrometry [M + Cl]- m / z is 519.3247 (cal. 519.2877, C 30 H 44 O5Cl); 1H-NMR and 13C-NMR data are shown in Table 1.

[0034] Table 1. 1H-NMR and 13C-NMR data of Compounds Ⅰ - Ⅳ

[0035]

[0036] The above results indicate that the structures of the obtained Compounds Ⅰ - Ⅶ are correct.

[0037] Example 2: Antitumor activities of Compounds Ⅰ - Ⅳ

[0038] 1) Experimental materials

[0039] Instruments and reagents: CO2 incubator (Jouan IGO150); fluorescence inverted microscope (Olympus IX51); MTT cell proliferation and cytotoxicity detection kit (Beyotime Biotechnology Research Institute), RPMI 1640 medium (GibcolBRL), RnaseA, fetal bovine serum, dimethyl sulfoxide (DMSO), trypsin (Shanghai Bioengineering Co., Ltd.).

[0040] Tumor cell lines for testing: Hela cells (human cervical cancer cells), K562 cells (human leukemia cells), K562 / ADR cells (human leukemia drug-resistant cells), SH-SY5Y cells (human neuroblastoma cells), Du-145 (human prostate cancer cells), HePG2 cells (human liver cancer cells), MCF-7 cells (human breast cancer cells), CT26 cells (colon cancer cells), purchased from the Cancer Institute of the Chinese Academy of Medical Sciences.

[0041] Test samples: Ursolic acid and the compounds of Formula I - Formula IV synthesized in Example 1, with a purity above 95%; meanwhile, cisplatin was selected as the positive control drug, and each compound was dissolved in DMSO and diluted.

[0042] 2) Experimental method

[0043] The MTT method was used to determine the half-maximal inhibitory concentration (IC 50 value) of each test compound against tumor cell lines: Tumor cells in the logarithmic growth phase were adjusted to a cell concentration of 5×10 5 / mL with RPMI 1640 culture medium containing 10% fetal bovine serum, inoculated into 96-well culture plates. The drug treatment group and the cell control group were added with 100 μL of cell suspension per well, and each group was set with 3 replicate wells. The blank control group was only added with 100 μL of RPMI 1640 complete culture medium per well, with 3 replicate wells. After culturing the 96-well culture plates 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 continued to culture for 72 h. According to the MTT method, the absorbance (A) value at 570 nm was measured with 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%]. The experiment was repeated 3 times. The regression equation was made using SPSS 11.5 software to calculate the half-maximal inhibitory concentration (IC 50 ) of each test sample against tumor cells after 72 h of action.

[0044] 3) Experimental results

[0045] According to the test results of the MTT method, the IC 50 values of the test samples against the above cells were calculated, and the results are shown in Table 2.

[0046] Table 2. Screening results of the in vitro cytotoxic activity of test samples

[0047]

[0048] The results show that the compounds shown in Formula I - Formula IV of the present invention have good anti-tumor activity and can be used as the active ingredients of anti-tumor drugs. At the same time, the compounds shown in Formula I - Formula IV of the present invention can effectively kill human leukemia drug-resistant cells.

[0049] 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 bearberry ketone acid derivative, characterized in that, The arbutinonic acid derivative is 3, 21-dioxo-12-ene-ursane-28-carboxylic acid, and the arbutinonic acid derivative has the structure of Formula I: 。 2. A bearberry quinone acid derivative, characterized in that, The arbutinonic acid derivative is 3, 21-dioxo-7 β -hydroxy-12-ene-ursane-28-carboxylic acid, and the arbutinonic acid derivative has the structure of formula II: 。 3. A bearberry quinone acid derivative, characterized in that, The said arbutinonic acid derivative is 3-oxo-21 β -hydroxy-11(12)-double bond-ursane-28, 13 β -lactone, and the said arbutinonic acid derivative has the structure of formula Ⅲ: 。 4. A bearberry quinone acid derivative, characterized in that, The arbutinonic acid derivative is 3-oxo-7 β , 21 β -dihydroxy-11(12)-double bond-ursane-28, 13 β -lactone, and the arbutinonic acid derivative has the structure of formula IV: 。 5. A method for preparing a bearberry quinone acid derivative by microbial transformation according to any one of claims 1-4, characterized in that, comprising the following steps: 1) Ferment and culture the microorganism, add arbutin to the culture medium, then carry out transformation culture, and obtain the fermentation broth after removing the mycelium. The microorganism is Aspergillus oryzae Aspergillus oryzae CGMCC 3.407; 2) Extracting the fermentation broth to obtain a transformant; 3) Purifying the transformant by reverse-phase silica gel column chromatography. The reverse-phase silica gel column purification is performed by gradient elution with a methanol-water two-phase system, and the fractions are collected and combined; 4) Purify the said components by reverse-phase high performance liquid chromatography to obtain ursolic acid derivatives, which include 3, 21-dioxo-12-ene-ursane-28-carboxylic acid, 3, 21-dioxo-7 β -hydroxy-12-ene-ursane-28-carboxylic acid, 3-oxo-21 β -hydroxy-11(12)-double bond-ursane-28, 13 β -lactone and 3-oxo-7 β , 21 β -dihydroxy-11(12)-double bond-ursane-28, 13 β -lactone.

6. Use of the arbutinonic acid derivative according to any one of claims 1-4 or the arbutinonic acid derivative prepared by the preparation method according to claim 5 in the preparation of an anti-tumor drug, wherein the tumor includes one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer, and colon cancer.

7. Use of the arbutinonic acid derivative according to any one of claims 1-4 or the arbutinonic acid derivative prepared by the preparation method according to claim 5 in the preparation of a drug for killing leukemia drug-resistant cells.

8. An anti-tumor drug, characterized in that, The active ingredient of the anti-tumor drug is the ursolic acid derivative or its pharmaceutically acceptable salt as described in any one of claims 1-4; the ursolic acid derivative is 3, 21-dioxo-12-ene-ursane-28-carboxylic acid, 3, 21-dioxo-7 β -hydroxy-12-ene-ursane-28-carboxylic acid, 3-oxo-21 β -hydroxy-11(12)-double bond-ursane-28, 13 β -lactone and 3-oxo-7 β , 21 β -dihydroxy-11(12)-double bond-ursane-28,13 β -lactone, or one or more of them; the tumors include one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

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