A kind of oleanane type triterpene derivative and its preparation method and application

Abstract

The present invention belongs to the field of medical technology, and discloses an oleanane-type triterpene derivative, a preparation method and an application thereof. The present invention utilizes microbial transformation technology to transform lupine-type triterpene betulinic acid into oleanane-type triterpene, and at the same time introduces a chemically reactive group into the inactive C-H bond of the oleanane-type triterpene mother nucleus. It is confirmed by an in vitro antitumor activity test that the oleanane-type triterpene derivative has significant antitumor activity and has a wide range of uses.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines and discloses an oleanane-type triterpene derivative and a preparation method and application thereof. Background Art

[0002] Betulinic acid, also known as lulutong acid and betulinic acid, belongs to the lupine-type pentacyclic triterpenoids and is widely distributed in medicinal plants, such as the bark of Betula platyphylla Suk. and the dried mature infructescence of Liquidambarformosana Hance (traditional Chinese medicine Lulutong). The content of betulinic acid in plants is generally low, so betulin or betulinic acid (betulinicaicd), which is more abundant in birch bark, is usually selected to obtain betulinic acid through chemical semi-synthesis or biotransformation.

[0003] Betulinic acid, as an oxidation product of betulinic acid, is an important intermediate in pharmaceutical chemistry research. Betulinic acid itself has a wide range of biological activities, such as anti-inflammatory, antibacterial, antiviral, antitumor, antioxidant, liver protection and other effects. However, the compound has a small polarity, is easily soluble in organic solvents, and is insoluble in water, resulting in low bioavailability. Chemical modification methods are usually used to carry out structural modification with betulinic acid as a substrate in order to obtain derivatives with stronger solubility and biological activity. In the prior art, due to the particularity of the structure of pentacyclic triterpenoid compounds, the parent nucleus lacks chemically reactive groups. The sites for structural modification of betulinic acid using organic chemical methods are mainly the two existing chemically reactive groups of betulinic acid-the carbonyl at position 3 and the carboxyl at position 28. Most of the CH bonds on the parent nucleus of betulinic acid are non-chemically reactive sites, and it is difficult to modify the parent nucleus structure using conventional chemical reaction methods, thereby obtaining derivatives modified with chemically reactive groups such as hydroxyl and carbonyl on the parent nucleus. Therefore, the diversity of reaction sites and derivatives of betulinic acid chemical structure modification is severely limited.

[0004] Microbial transformation is the process of using enzymes in microbial cells to modify the structure of substrates and transform them into other compounds. The special intracellular or extracellular enzymes produced by fermentative fungi, actinomycetes and other microorganisms during their growth can not only catalyze their own substances, but also catalyze specific chemical reactions for foreign compounds, such as hydrolysis, oxidation, reduction, cleavage, skeleton rearrangement, etc., thus becoming a powerful tool for structural modification and transformation of complex natural products. Our research group has long been engaged in the research of microbial transformation of natural active ingredients, especially the microbial transformation of natural active triterpenes. Using a variety of microorganisms to transform common triterpenes from natural sources (including dammarane-type, cycloartane-type, ursane-type, oleanane-type triterpenes and lupetane-type, etc.), it was found that the microbial enzyme system can selectively catalyze multiple non-chemical reaction active sites on the triterpene mother nucleus, thereby obtaining derivatives with chemically active groups such as hydroxyl and carbonyl groups on the mother nucleus (Journal of natural products 2021, 84: 2664-2674; Phytochemistry 2021, 182: 112608; Natural Product Research 2021, 35(16): 2685-2690; Phytochemistry 2019, 166: 112076; Planta Medica 2019, 85(1): 56-61). On the one hand, microbial transformation products can obtain derivatives with stronger biological activity that can be directly used in drug research and development. On the other hand, the newly introduced chemically active 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 organic chemical preparation of triterpenoid derivatives.

[0005] At present, the research on natural active triterpenoids in organic chemistry mainly focuses on the preparation of derivatives through chemical reactions such as esterification, acylation, and heterocyclization, and there are few studies on the mutual conversion between different types of triterpenoids. Liu et al. reported the use of organic chemical methods to convert lupane-type triterpenes into oleanane-type triterpenes. After three steps of reaction, lupane-type triterpenoid 23-hydroxybetulinic acid (2) can be converted into oleanane-type triterpenoid 3,23-dihydroxyoleanane-28,19-lactone (Fitoterapia 2015, 100: 50-55). Although this chemical method can convert lupane-type triterpenes into oleanane-type triterpenes, it cannot activate the non-chemically reactive sites of the parent nucleus and introduce chemically reactive groups.

[0006]

[0007] Unlike organic chemistry methods, microbial transformation cannot predict or determine the transformation ability of microorganisms theoretically or by the fermentation method itself, nor can it predict or determine the structure of the transformation product. It can only be confirmed after the compound is prepared and then identified by structure. Studies have shown that even for the same lead compound, the derivatives obtained after selective catalysis by different types of microbial enzyme systems will have very significant differences in structure. This is also the product diversity feature of microbial transformation. Therefore, as a method for structural modification of organic compounds, the product structure of microbial transformation has the characteristics of diversity and uncertainty. It can only be finally determined after preparation and structural confirmation. Therefore, microbial transformation cannot design compounds first and then reverse synthesize them like organic chemistry. At the same time, there are many types of microorganisms in nature, and the transformation abilities of different types of microorganisms vary greatly. Selecting suitable microbial strains is the key to structural modification and transformation of asiatic acid using microbial transformation. As a complete compound structure, the drug molecule is only different in carbonyl, hydroxyl and methyl groups, and the pharmacological activity it exhibits will also be significantly different. Before the corresponding compound is obtained and pharmacological tests are performed, its effect cannot be accurately predicted. Therefore, the screening and selection of microbial strains are crucial for discovering compounds with better activity. Summary of the invention

[0008] In view of this, the object of the present invention is to provide an oleanane-type triterpene derivative or a pharmaceutically acceptable salt thereof and a preparation method thereof, wherein the oleanane-type triterpene derivative can be used to prepare an anti-tumor drug.

[0009] The oleanane-type triterpene derivative provided by the present invention is a compound having the structural formula I:

[0010]

[0011] The compound with the structural formula I is a new oleanane-type triterpene derivative disclosed for the first time in the present invention.

[0012] The present invention also provides a method for preparing the above-mentioned oleanane-type triterpene derivatives, comprising the following steps:

[0013] 1) fermenting and culturing a microorganism, adding betulinic acid to a culture medium, then performing transformation culture, removing mycelium to obtain a fermentation liquid, wherein the microorganism is a strain of the genus Cichorium;

[0014] 2) extracting the fermentation broth, and evaporating the extract to obtain a crude conversion extract;

[0015] 3) The crude extract was subjected to silica gel column chromatography with dichloromethane:methanol as the mobile phase for gradient elution. The fractions were collected and analyzed by TLC to obtain 5 components.

[0016] 4) Purifying the components by reverse phase high performance liquid chromatography to obtain oleanane type triterpene derivatives.

[0017] Preferably, the microorganism is Circinella muscae CGMCC 3.2695.

[0018] Preferably, in step 1), the transformation culture temperature is 30° C., the shaking speed is 130 rpm, and the culture time is 120 hours.

[0019] Preferably, in step 2), the extraction solvent is dichloromethane.

[0020] Preferably, in step 3), the gradient elution conditions are dichloromethane:methanol 100:0-50:1-30:1-20:1-10:1.

[0021] Compared with the prior art, the present invention utilizes microbial transformation technology to convert lupeane-type triterpene betulinic acid into oleanane-type triterpene, and simultaneously successfully performs structural modifications on non-chemically reactive sites 7, 15 and 29 on the parent nucleus, and introduces chemically reactive groups hydroxyl groups, thereby obtaining a novel oleanane-type triterpene derivative compound of formula I. In vitro anti-tumor cell tests have confirmed that compound of formula I has significant anti-tumor activity, can be used as an active ingredient in anti-tumor drugs, and has a wide range of uses.

[0022] DETAILED DESCRIPTION

[0023] In order to further illustrate the present invention, a novel oleanane-type triterpene derivative and a preparation method thereof provided by the present invention are described in detail below in conjunction with examples.

[0024] Example 1: Preparation of a compound having the structural formula I

[0025] The present invention adopts a microbial transformation method, using betulinic acid as a raw material, and preparing the compound of the present invention through fermentation, extraction, separation and other steps. The strain of Circinella can be purchased from the Microbial Culture Collection Management Center of the Chinese Academy of Sciences (CGMCC), and a potato culture medium is selected and stored in a refrigerator at 4°C on a solid slant culture medium.

[0026] Taking Circinella muscae CGMCC 3.2695 as an example, the process for preparing the compound of formula I is as follows:

[0027] 1) Fermentation, transformation and extraction

[0028] Circinella muscae CGMCC 3.2695 was inoculated into two 250 mL triangular flasks (containing 100 mL potato culture medium) as seed liquid. After 1 day of shaking culture at 130 rpm and 30 ° C on a shaker, when the mycelium is in a vigorous growth period, 1 mL of seed liquid was taken with a sterile pipette and added to 20 1000 mL shake flasks (containing 400 mL potato culture medium). After 1 day of shaking culture, 20 mg of betulinic acid (0.2 mL, 100 mg / mL DMSO solution) was added to each shake flask, and 400 mg of substrate was used in total. The transformation was continued for 5 days under the same conditions, the fermentation broth was filtered, the mycelium was filtered out, and the filtrate was extracted 3 times with an equal volume of dichloromethane. The extract was concentrated to dryness under reduced pressure to obtain a crude extract of about 0.52 g of the transformant.

[0029] 2) Silica gel column chromatography separation

[0030] The crude extract was separated by silica gel column chromatography using a gradient elution of dichloromethane:methanol (100:0, 50:1, 30:1, 20:1, 10:1). The fractions were collected and combined after TLC analysis to obtain combined components AE.

[0031] 3) Reverse-phase HPLC purification

[0032] The combined fraction C was purified by reverse phase high performance liquid chromatography. The preparation conditions were semi-preparative chromatography column YMC ODS A-5μm, 10.0×250mm, acetonitrile-water (55:45, V / V), flow rate 2.5mL / min, detection wavelength 203nm. The conversion product with the structural formula of formula I was obtained, and its mass spectrum and spectral data are shown below.

[0033] Compound I: 3-oxo-7β,15α,29-trihydroxyoleanan-28β,19β-lactone; melting point 304–306℃; optical rotation :+37.5°(c=0.1,MeOH); Main absorption peak of infrared spectrum (KBr)ν max :3551,3032,2968,1734,1695,1381,1226,1048cm -1 ; High resolution mass spectrum m / z 525.3192[M+Na] + (calcd.525.3187for C 30 H 46 O6Na); the H NMR and C NMR data are shown in Table 1.

[0034] Table 1. Attribution of H-NMR and C-NMR data of compound Ⅰ (deuterated chloroform)

[0035]

[0036]

[0037] The above results show that the structure of the obtained compound is correct.

[0038] Example 2: Antitumor activity of compound I

[0039] 1) Experimental Materials

[0040] Instruments and reagents: CO2 incubator (Jouan IGO150); microplate reader (Bio-TEK ELx800); fluorescence inverted microscope (Olympus IX51); MTT cell proliferation and cytotoxicity detection kit (Biyuntian Biotechnology Research Institute), RPMI 1640 medium (Gibcol BRL), RNase A, fetal bovine serum, dimethyl sulfoxide (DMSO), trypsin (Shanghai Bioengineering Co., Ltd.).

[0041] The tumor cell lines used for the test: Hela cells (human cervical cancer cells), K562 cells (human leukemia cells), K562 / ADR cells (human leukemia 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), were purchased from the Institute of Oncology, Chinese Academy of Medical Sciences.

[0042] Test samples: betulinic acid and compound I synthesized in Example 1, with a purity of more than 95%; at the same time, cisplatin was selected as a positive control drug, and each compound was dissolved in DMSO and then diluted.

[0043] 2) Experimental methods

[0044] The MTT method was used to determine the half inhibition rate IC of each test compound on tumor cell lines. 50 Value: Take tumor cells in the logarithmic growth phase and adjust the cell concentration to 5×10 5 / mL, inoculated in 96-well culture plates, 100 μL of cell suspension was added to each well of the drug-treated group and the cell control group, and 3 replicates were set up for each group. Only RPM I 1640 complete culture medium was added to the blank control group, 100 μL per well, and 3 replicates were set up. After the 96-well culture plate was placed in a 37°C, 5% CO2 incubator for 24 hours, different concentrations of the test samples were added to make the final concentration 0.1-100 μM, and the culture was continued for 72 hours. The absorbance (A) value at 570 nm was measured on a microplate reader according to the MTT method, and the inhibition rate was calculated [inhibition rate = (1-experimental group A value / control group A value) × 100%]. The experiment was repeated 3 times. SPSS 11.5 software was used to make a regression equation to calculate the half-maximal inhibitory concentration (IC) of each test sample on tumor cells for 72 hours. 50 ).

[0045] 3) Experimental results

[0046] According to the MTT test results, calculate the IC of the test sample for the above cells 50 The results are shown in Table 2.

[0047] Table 2. In vitro cytotoxic activity screening results of test samples

[0048]

[0049] The results show that the compound I of the present invention has good anti-tumor activity and can be used as an active ingredient of anti-tumor drugs.

[0050] Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. An oleanane-type triterpene derivative, characterized in that: The oleanane-type triterpene derivative is 3-carbonyl-7β,15α,29-trihydroxyoleanane-28β,19β-lactone, having a structure of Formula I:

2. A method for preparing the oleanane-type triterpene derivative according to claim 1, characterized in that: The method is prepared by using betulinic acid as a substrate and a microbial transformation method, comprising the following steps: 1) fermenting and culturing a microorganism, adding betulinic acid to a culture medium, then performing transformation culture, removing mycelium to obtain a fermentation liquid, wherein the microorganism is Circinella muscae CGMCC 3.2695; the transformation culture temperature is 30° C., the shaking speed is 130 rpm, and the culture time is 120 hours; 2) extracting the fermentation broth to obtain a transformant; 3) purifying the transformed product by column chromatography, wherein the silica gel column purification adopts a dichloromethane-methanol two-phase system gradient elution, and collecting and merging the components; 4) Purifying the components by reverse phase high performance liquid chromatography to obtain 3-carbonyl-7β,15α,29-trihydroxyoleanane-28β,19β-lactone.

3. Use of the oleanane-type triterpene derivative according to claim 1 in the preparation of antitumor drugs.

4. The use according to claim 3, characterized in that: The tumor is one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

5. An anti-tumor drug, characterized in that: The active ingredient of the anti-tumor drug is the oleanane-type triterpene derivative according to claim 1, and the tumor is one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

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