Ursane-type triterpenoid compounds, microbial conversion preparation methods and applications thereof

Abstract

The present invention belongs to the field of medical technology and discloses an ursane-type triterpenoid compound, a microbial conversion preparation method and application thereof. The ursane-type triterpenoid compound has both an 11β, 12β-three-membered oxygen ring and a 28, 13β-lactone. The present invention utilizes the enzyme system of a microorganism and uses ursolic acid as a substrate to specifically introduce a three-membered oxygen ring at the 11β, 12β positions of the ursane-type triterpenoid mother nucleus and a lactone at the 28, 13β positions. In addition, hydroxyl groups or double bonds are successfully introduced into the non-chemically reactive sites 1, 7 and 21 of the ursane-type triterpenoid mother nucleus to obtain seven ursane-type triterpenoid compounds with novel structures. The seven compounds are confirmed by in vitro anti-tumor cell tests to have significant anti-tumor activity and can be used as active ingredients of anti-tumor drugs with a wide range of medical uses.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to an ursane-type triterpene compound, a microbial conversion preparation method and application thereof, and in particular to a preparation method of a novel ursane-type triterpene having both an 11β, 12β-three-membered oxygen ring and a 28, 13β-lactone, and application thereof in the preparation of anti-tumor drugs. Background Art

[0002] About 25% of the global drug market comes from plant secondary metabolites, of which the largest number and types are terpene hydrocarbon compounds and their oxygen-containing derivatives (terpenes). According to the number of isoprene units (C5H8) in their structure, they can be divided into monoterpenes (C 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), triterpenes (C 30 ) etc. Malignant tumors are one of the most common diseases today. With the accelerated pace of life and increased stress, the incidence of malignant tumors is increasing year by year. Therefore, actively searching for natural medicines has important clinical value in anti-tumor treatment.

[0003] Natural triterpenoids have over 100 different skeleton types. In recent years, increasing research has focused on the bioactivity of ursane-type triterpenes in areas such as antibacterial, anti-inflammatory, anti-tumor, cytotoxic, and liver protection. However, the highly hydrophobic nature of triterpenoid molecules significantly limits their effective clinical use as pharmaceuticals. Currently, one of the most common approaches to improving the bioavailability of triterpenoids is chemical modification. Due to the unique structure of pentacyclic triterpenes, the core lacks chemically reactive groups. Organic chemical methods primarily target two common chemically reactive groups in the structure for structural modification of ursane-type triterpenes: the hydroxyl group at position 3 on the A ring and the carboxyl group at position 28 on the D / E ring. Most C-H bonds in the core of ursane-type triterpenes are chemically inactive, making it difficult to modify the core structure using conventional chemical reaction methods to obtain derivatives modified with chemically reactive groups such as hydroxyl or carbonyl groups. This severely limits the diversity of chemically modified sites and derivatives of ursane-type triterpenes. Another approach is biotransformation, which utilizes mild conditions and the highly regio- and stereoselective catalytic activity of microorganisms. This makes it a powerful tool for structural modification and engineering of complex natural products. Our research group has long been engaged in research on the microbial transformation of natural active ingredients, particularly triterpenes. A variety of microorganisms were used to study the transformation of common triterpenes from natural sources (including dammarane-type, cycloaltinane-type, ursane-type, oleanane-type triterpenes and lupeane-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 for drug development. On the other hand, the newly introduced chemically active groups on the mother 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 derivatives of triterpenoid compounds.

[0004] However, unlike chemical synthesis methods for structural modification and transformation of organic compounds, microbial transformation methods cannot theoretically predict or determine which microorganisms possess the ability to transform, nor can the chemical structure of the transformation products be predicted or determined, either through the fermentation process itself or theoretically. Confirmation of the chemical structure of the transformation products can only be achieved after obtaining the transformation products and undergoing structural characterization. Previous studies have shown that even for structurally similar substrates, the structures of the transformation products obtained after selective catalysis by microbial enzyme systems can differ significantly. For example, betulinic acid and betulinic acid differ structurally only in the hydroxyl and carbonyl groups at the C-3 position, yet their derivatives obtained after microbial transformation show significant differences (Journal of Natural Products 2021, 84:2664-2674; Phytochemistry 2021, 182:11-2608). Furthermore, even for the same lead compound, the structures of the derivatives obtained after catalysis by different microbial enzymes can vary significantly, a characteristic of the diversity of microbial transformation. Therefore, as a method for structural modification of organic compounds, microbial transformation methods are characterized by diversity and uncertainty in their product structures, which can only be determined after preparation and structural characterization. Therefore, microbial transformation methods cannot be compared to chemical synthesis methods, where a compound is designed first and then reverse-synthesized. Furthermore, the diversity of microorganisms in nature makes selecting the right microbial strain crucial for structural modification and transformation of compounds using microbial transformation methods. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a novel ursane-type triterpenoid compound having both an 11β,12β-three-membered oxygen ring and a 28,13β-lactone or a pharmaceutically acceptable salt thereof and a preparation method thereof, wherein the novel ursane-type triterpenoid compound can be used to prepare anti-tumor drugs.

[0006] The novel ursane-type triterpenoid compounds provided by the present invention are compounds having the structural formulas of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI and Formula VII:

[0007]

[0008] The compounds with the structural formulas of formula I to formula VII are all novel ursane-type triterpenoid compounds disclosed for the first time in the present invention.

[0009] The present invention also provides a method for preparing the novel ursane-type triterpenoid compound, comprising the following steps:

[0010] 1) fermenting and culturing a microorganism, adding ursolic acid to a culture medium, then performing a transformation culture, and removing mycelium to obtain a fermentation liquid, wherein the microorganism is a strain of the genus Aspergillus;

[0011] 2) extracting the fermentation broth and evaporating the extract to obtain a crude conversion extract;

[0012] 3) The crude extract was subjected to silica gel column chromatography with dichloromethane:methanol as the mobile phase for gradient elution. The collected fractions were analyzed by HPLC and combined to obtain five components.

[0013] 4) Purifying the components by reverse-phase high performance liquid chromatography to obtain novel ursane-type triterpenoid compounds.

[0014] The synthetic route of the above-mentioned preparation method is as follows:

[0015]

[0016] Preferably, in step 1), the transformation culture temperature is 26° C., the shaking speed is 160 rpm, and the culture time is 168 hours.

[0017] Preferably, in step 2), the extraction solvent is ethyl acetate.

[0018] Preferably, in step 3), the gradient elution condition is dichloromethane:methanol 100:1-80:1-60:1-50:1-20:1.

[0019] The present invention also provides the use of the above-mentioned ursane-type triterpenoid compounds in the preparation of anti-tumor drugs, wherein the tumor is one or more of cervical cancer, leukemia, neuroblastoma, prostate cancer cells, liver cancer, breast cancer and colon cancer.

[0020] Compared to existing technologies, the present invention utilizes a microbial transformation method to specifically and simultaneously introduce a three-membered oxygen ring at the 11β and 12β positions of the ursane-type triterpene nucleus, and a lactone at the 28 and 13β positions. These introduced groups are all stereoselective. Furthermore, the present invention successfully structurally modifies the non-chemically reactive sites 1, 7, and 21 of the ursane-type triterpene nucleus, introducing chemically reactive groups such as hydroxyl groups or double bonds. This yields novel structural ursane-type triterpene compounds (Formulas I to VII). In vitro anti-tumor cell assays confirm that compounds I to VII exhibit significant anti-tumor activity and can be used as active ingredients in anti-tumor drugs or as intermediates in chemical synthesis, with a wide range of applications. DETAILED DESCRIPTION

[0021] To further illustrate the present invention, the novel ursane-type triterpenes and their preparation methods provided by the present invention are described in detail below with reference to the examples.

[0022] Example 1: Preparation of compounds of formula I to formula VII

[0023] The present invention adopts a microbial transformation method, using ursolic acid as a raw material, and prepares the compound of the present invention through fermentation, extraction, separation and other steps. Aspergillus strains can be purchased from the China General Collection of Microorganisms (CGMCC) of the Chinese Academy of Sciences. Potato culture medium is used, and the culture medium is stored on a solid slant medium in a refrigerator at 4°C.

[0024] Taking Aspergillus ochraceus CGMCC3.5324 as an example, the process for preparing compounds of formula I to formula VII is as follows:

[0025] 1) Fermentation, conversion and extraction

[0026] Aspergillus ochraceus CGMCC3.5324 was inoculated into two 250 mL Erlenmeyer flasks (filled with 100 mL of potato culture medium) as seed culture. After shaking on a shaker at 160 rpm and 26°C for 12 hours, once mycelial growth reached a vigorous stage, 1 mL of seed culture was pipetted using a sterile pipette and added to 20 1000 mL shake flasks (filled with 400 mL of potato culture medium). After shaking for 24 hours, 20 mg of ursolic acid (0.2 mL of a 100 mg / mL ethanol solution) was added to each shake flask, for a total of 400 mg of substrate. Transformation was continued under the same conditions for three days. The fermentation broth was filtered to remove mycelium, and the filtrate was extracted three times with equal volumes of ethyl acetate. The extract was then concentrated to dryness under reduced pressure to yield approximately 0.85 g of crude transformant extract.

[0027] 2) Silica gel column chromatography separation

[0028] The crude extract was separated by silica gel column chromatography using a dichloromethane:methanol gradient elution (100:1-80:1-60:1-50:1-20:1). The collected fractions were combined and analyzed by HPLC to obtain combined fractions AE.

[0029] 3) Reverse-phase high performance liquid chromatography purification

[0030] Combined fraction B was purified by reverse-phase HPLC. Preparation conditions were: a semi-preparative column YMCODSA-5μm, 10.0×250mm, acetonitrile-water (45:55, V / V), a flow rate of 3.0 mL / min, and a detection wavelength of 203 nm. Conversion products with the structural formulae of Formula I, Formula II, and Formula III were obtained. Combined fraction C was purified by reverse-phase HPLC. Preparation conditions were: a semi-preparative column YMCODSA-5μm, 10.0×250mm, acetonitrile-water (60:40, V / V), a flow rate of 3.0 mL / min, and a detection wavelength of 203 nm. Conversion products with the structural formulae of Formula IV and Formula V were obtained. Combined fraction D was purified by reverse-phase HPLC. Preparation conditions used a semi-preparative column, YMCODSA-5 μm, 10.0 × 250 mm, acetonitrile-water (66:34, V / V), a flow rate of 3.0 mL / min, and a detection wavelength of 203 nm. Conversion products with the structural formulae VI and VII were obtained. The mass spectra and spectroscopic data for compounds of Formulas I to VII are shown below.

[0031] Compound I: 3-oxo-21β-hydroxyl-11β,12β-epoxy-urs-28,13β-olide: melting point 257–263°C; optical rotation The main absorption peak of infrared spectrum (KBr):ν max 3612,2943,1765,1718,1377,1226,1048cm -1 ; High-resolution mass spectrometry m / z 529.3173 [M+COOH] - (calcd.forC 31 H 45 O7,529.3165); H NMR and C NMR data are shown in Table 1.

[0032] Compound II: 3-oxo-1β,21β-dihydroxyl-11β,12β-epoxyl-urs-28,13β-olide: melting point 278–280°C; optical rotation The main absorption peak of infrared spectrum (KBr):ν max 3668,3527,2961,1768,1722,1362,1239,1054cm -1 ; High-resolution mass spectrometry m / z 545.3156 [M+COOH] - (calcd.forC31 H 45 O8,545.3114); H NMR and C NMR data are shown in Table 1.

[0033] Compound III: 3-oxo-21β-hydroxyl-11β,12β-epoxy-1(2)-double bond-ursane-28,13β-lactone (3-oxo-21β-hydroxyl-11β,12β-epoxyl-urs-1-ene-28,13β-olide): melting point 242–245°C; optical rotation The main absorption peak of infrared spectrum (KBr):ν max 3622,3031,2955,1765,1689,1332,1267,1091cm -1 ; High-resolution mass spectrometry m / z 527.2993 [M+COOH] - (calcd.forC 31 H 43 O7,527.3009); H NMR and C NMR data are shown in Table 1.

[0034] Compound IV: 3-oxo-7α,21β-dihydroxyl-11β,12β-epoxy-urs-28,13β-olide: melting point 270–274°C; optical rotation The main absorption peak of infrared spectrum (KBr):ν max 3685,3618,2956,1767,1717,1322,1247,1053cm -1 ; High-resolution mass spectrometry m / z 545.3130 [M+COOH] - (calcd.forC 31 H 45 O8,545.3114); H NMR and C NMR data are shown in Table 2.

[0035] Compound V: 3-oxo-7β,21β-dihydroxyl-11β,12β-epoxy-urs-28,13β-olide: melting point 268–273°C; optical rotation The main absorption peak of infrared spectrum (KBr):ν max3647,3586,2959,1765,1713,1351,1232,1055cm -1 ; High-resolution mass spectrometry m / z 545.3123 [M+COOH] - (calcd.forC 31 H 45 O8,545.3114); H NMR and C NMR data are shown in Table 2.

[0036] Compound VI: 3,21-dioxo-7β-hydroxyl-11β,12β-epoxy-urs-28,13β-olide: melting point 247–249°C; optical rotation The main absorption peak of infrared spectrum (KBr):ν max 3674,2956,1765,1721,1714,1366,1267,1068cm -1 ; High-resolution mass spectrometry m / z 543.2968 [M+COOH] - (calcd.forC 31 H 43 O8,543.2958); the H NMR and C NMR data are shown in Table 2.

[0037] Compound VII: 3-oxo-21β-hydroxyl-7-methyl-11β,12β-epoxy-7-double bond-26-norurs-28,13β-olide: melting point 282–284°C; optical rotation The main absorption peak of infrared spectrum (KBr):ν max 3598,2971,1767,1716,1380,1233,1048cm -1 ; High-resolution mass spectrometry m / z 527.3017 [M+COOH] - (calcd.forC 31 H 43 O7,527.3009); H NMR and C NMR data are shown in Table 3.

[0038] Table 1. H-NMR and C-NMR data of compounds I-III

[0039]

[0040]

[0041] Table 2. H-NMR and C-NMR data of compounds IV-VI

[0042]

[0043] Table 3. H NMR and C NMR data of compound VII

[0044]

[0045]

[0046] The above results show that the structures of the obtained compounds of formula I to formula VII are correct.

[0047] Example 2: Antitumor activity of compounds of formula I to formula VII

[0048] 1) Experimental Materials

[0049] Instruments and reagents: CO2 incubator (Jouan IGO150); microplate reader (Bio-TEKEL x800); fluorescence inverted microscope (Olympus IX51); MTT cell proliferation and cytotoxicity detection kit (Biyuntian Biotechnology Research Institute), RPMI1640 medium (Gibcol BRL), RNase A, fetal bovine serum, dimethyl sulfoxide (DMSO), and trypsin (Shanghai Bioengineering Co., Ltd.).

[0050] The tumor cell lines used in 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.

[0051] Test samples: ursolic acid and the compounds of formula I to formula VII 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.

[0052] 2) Experimental methods

[0053] The MTT method was used to determine the half inhibition rate IC of each test compound on tumor cell lines. 50 Value: Tumor cells in the logarithmic growth phase were taken and the cell concentration was adjusted to 5×10 5 / mL, seeded 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 RPMI1640 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 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-A value of the experimental group / A value of the control group) × 100%]. The experiment was repeated 3 times. SPSS11.5 software was used to make a regression equation to calculate the half-maximal inhibitory concentration (IC50) of each test sample on tumor cells for 72 hours. 50 ).

[0054] 3) Experimental results

[0055] According to the MTT test results, calculate the IC value of the test sample for the above cells. 50 The results are shown in Table 2.

[0056] Table 2. In vitro cytotoxic activity screening results of test samples

[0057]

[0058] The results show that the compounds of formula I to formula VII of the present invention have good anti-tumor activity and can be used as active ingredients of anti-tumor drugs and applied to the preparation of anti-tumor drugs.

[0059] Those skilled in the art will appreciate that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An ursane-type triterpenoid compound, characterized in that: The ursane-type triterpenoid compound has a structure of formula I: 。 2. An ursane-type triterpenoid compound, characterized in that: The ursane-type triterpenoid compound has a structure of formula II: 。 3. An ursane-type triterpenoid compound, characterized in that: The ursane-type triterpenoid compound has a structure of formula III: 。 4. An ursane-type triterpenoid compound, characterized in that: The ursane-type triterpenoid compound has a structure of formula IV: 。 5. An ursane-type triterpenoid compound, characterized in that: The ursane-type triterpenoid compound has a structure of formula V: 。 6. An ursane-type triterpenoid compound, characterized in that: The ursane-type triterpenoid compound has a structure of formula VI: 。 7. An ursane-type triterpenoid compound, characterized in that: The ursane-type triterpenoid compound has a structure of formula VII: 。 8. A method for preparing ursane-type triterpenoid compounds by microbial conversion, characterized in that: The steps include: 1) fermenting and culturing a microorganism, adding ursolic acid to a culture medium, then performing transformation culture, and removing mycelium to obtain a fermentation broth, wherein the microorganism is Aspergillus ochraceus CGMCC 3.5324; 2) extracting the fermentation broth to obtain a transformant; 3) purifying the transformed product by silica gel column chromatography using a dichloromethane-methanol two-phase system gradient elution, and collecting and combining the fractions; 4) purifying the components by reverse-phase high performance liquid chromatography to obtain ursane-type triterpenoid compounds of formula I to formula VII; 。 9. The preparation method according to claim 8, characterized in that The extraction solvent of the extraction is ethyl acetate.

10. Use of the ursane-type triterpenoid compound according to any one of claims 1 to 7 in the preparation of an anti-tumor drug, wherein the tumor is one or more of cervical cancer, leukemia, neuroblastoma, prostate cancer cell, liver cancer, breast cancer and colon cancer.

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