13,28-epoxy-substituted ushane-type triterpenes, methods for their preparation and use

Abstract

The application belongs to the technical field of medicine, and discloses a kind of 13, 28-epoxy substituted ussane type triterpenes and its preparation method and application. The application uses microbial transformation technology, with ursolic acid as substrate, and introduces epoxy group substitution in 13 and 28 positions with regional selectivity and stereoselectivity, and introduces hydroxyl, carbonyl or acetoxy in multiple positions of the parent nucleus, so that the novel structure of 13, 28-epoxy substituted ussane type triterpene derivatives formula I-IX is obtained. And through in vitro anti-tumor cell test, it is confirmed that compounds formula I-IX have significant anti-tumor activity, can be used as active ingredients of antitumor drugs, and have wide application.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a 13,28-epoxy-substituted ursane-type triterpenoid, its preparation method, and its application. Background Technology

[0002] Ursine-type triterpenes, also known as α-amyrin-type triterpenes, are a class of terpenoid compounds whose basic parent nucleus consists of 30 carbon atoms. Most of these triterpenes are derivatives of ursine, with their A / B, B / C, and C / D rings all in a trans configuration, and their D / E ring in a cis configuration. They mainly exist in free or glycosidic forms in plants of the Rosaceae, Caprifoliaceae, and Oleaceae families, such as Rosa laevigata root, Ilex chinensis, and Centella asiatica. These include compounds such as α-amyrin, ursolic acid, and taraxasterol. Glycosides generally involve the substitution of the hydroxyl group at position 3 and the carboxyl group at position 28, with the substituted monosaccharides mostly being glucose, arabinose, rhamnose, and glucuronic acid. Modern pharmacological studies have shown that these compounds possess antitumor, antioxidant, anti-inflammatory, antibacterial, hypoglycemic, and hepatoprotective activities, and they are widely used in the pharmaceutical and cosmetic fields. Currently, the main method for obtaining these triterpenoid compounds is direct extraction and isolation from plants, followed by chemical synthesis or semi-synthesis to obtain their derivatives.

[0003] However, direct extraction and isolation from plants is limited by the availability of natural resources. Chemical synthesis and semi-synthetic terpenoids, due to their complex structures, multiple stereocenters, limited number of hydrogen bond donors in the parent nucleus, and limited number of rotatable bonds in the rigid framework, restrict further chemical biological research. In recent years, with the rise of synthetic biology, microbial transformation has become a simple and effective technique for modifying the structure of natural compounds. It can introduce functional groups regioselectively and stereoselectively, offering advantages such as high efficiency, economy, and environmental friendliness—advantages difficult to achieve with conventional chemical modification. However, compared to chemical synthesis, microbial transformation is highly random; the results cannot be predicted as accurately as in chemical synthesis, nor can they be designed purposefully. Furthermore, even similar substrates transformed by the same or different strains can yield significantly different products. Conversely, the same substrate transformed by different strains may also yield significantly different products. Therefore, selecting appropriate strains is crucial when using microbial transformation for compound structure modification. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a method for preparing 13,18-epoxy-substituted ursane-type triterpenes or pharmaceutically formable salts thereof and their microbial transformation, wherein these ursane-type triterpenes can be used to prepare antitumor drugs.

[0005] The 13,18-epoxy-substituted ursane-type triterpenes provided by this invention are 3β,7β,15α,21β-tetrahydroxy-13β,28β-epoxy-10-double-bond-ursane, 3β,15α,21β-trihydroxy-13β,28β-epoxy-10-double-bond-ursane, 3β,7β-dihydroxy-21β-acetoxy-13β,28β-epoxy-10-double-bond-ursane, 7β,15α,21β-trihydroxy-3β-acetoxy-13β,28β-epoxy-10-double-bond-ursane, and 7β,21β-dihydroxy-3-carbonyl-13β,28β-cyclo Oxy-10-double bond-ursane, 3β,7β,21β-trihydroxy-28-methoxy-13β,28β-epoxy-10-double bond-ursane, 3β,7β,15α,21β,23-pentahydroxy-13β,28β-epoxy-10-double bond-ursane, 3β,7β,21β,24-tetrahydroxy-13β,28β-epoxy-10-double bond-ursane, and 3β,7β,24-trihydroxy-21-carbonyl-13β,28β-epoxy-10-double bond-ursane, with structures shown in Formulas I, II, III, IV, V, VI, VII, VIII, and IX, respectively:

[0006]

[0007] All compounds with structural formulas I through IX are novel ursane-type triterpenes disclosed for the first time in this invention.

[0008] This invention also provides a method for preparing the above-mentioned novel ursane-type triterpenoids by microbial transformation, comprising the following steps:

[0009] 1) Fermentation culture of microorganisms: Arbutin was added to the culture medium, followed by transformation culture. After removing the mycelium, the fermentation broth was obtained. The microorganisms were Actinomucor elegans CGMCC 3.3393, Mucor erectus CGMCC 3.180, and Mucor racemosus CGMCC 3.20.

[0010] 2) The fermentation broth is extracted to obtain the transformed product;

[0011] 3) The transformed product was purified by reversed-phase silica gel column chromatography using a methanol-water two-phase system with gradient elution, and the fractions were collected and combined.

[0012] 4) The components were purified by reversed-phase high-performance liquid chromatography to obtain 3β,7β,15α,21β-tetrahydroxy-13β,28β-epoxy-10-double bond-ursane, 3β,15α,21β-trihydroxy-13β,28β-epoxy-10-double bond-ursane, 3β,7β-dihydroxy-21β-acetoxy-13β,28β-epoxy-10-double bond-ursane, 7β,15α,21β-trihydroxy-3β-acetoxy-13β,28β-epoxy-10-double bond-ursane, and 7β,21β-dihydroxy-13β,28β-epoxy-10-double bond-ursane. The compounds are 3β-carbonyl-13β,28β-epoxy-10-double bond-ursane, 3β,7β,21β-trihydroxy-28-methoxy-13β,28β-epoxy-10-double bond-ursane, 3β,7β,15α,21β,23-pentahydroxy-13β,28β-epoxy-10-double bond-ursane, 3β,7β,21β,24-tetrahydroxy-13β,28β-epoxy-10-double bond-ursane, and 3β,7β,24-trihydroxy-21-carbonyl-13β,28β-epoxy-10-double bond-ursane.

[0013] The present invention also provides the application of the above-mentioned novel ursane-type triterpenoids in the preparation of antitumor drugs, wherein the tumors include one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer, and colon cancer.

[0014] Compared with existing technologies, this invention utilizes the catalytic action of microbial enzymes to form epoxy groups at the 13β and 28β positions of ursane with regioselectivity and stereoselectivity, while simultaneously introducing hydroxyl, carbonyl, or acetoxy groups at the 7β, 15α, 21β, 23, or 24 positions. This yields a novel 13,28-epoxy-substituted ursane-type triterpenoid, formula I-IX. In vitro antitumor cell assays have confirmed that compound I-IX possesses significant antitumor activity and can serve as an active ingredient in antitumor drugs, demonstrating broad applicability. Detailed Implementation

[0015] To further illustrate the present invention, the following describes in detail, with reference to embodiments, the preparation method of the novel 13,28-epoxy-substituted ursane-type triterpenoid provided by the present invention and its application in the preparation of antitumor drugs.

[0016] Example 1: Preparation of novel ursane-type triterpenes with structural formulas I-IX

[0017] This invention employs a microbial transformation method, using arbutin as a substrate, to prepare the compounds of this invention through steps such as biotransformation, extraction, and separation. Taking *Actinomucor elegans* CGMCC 3.3393 as an example, the process for preparing compounds with structural formulas I through IX is as follows:

[0018] 1) Microbial transformation

[0019] Preparation of seed culture: Pour the autoclaved PDB medium into two 250mL Erlenmeyer flasks, each containing 100mL of medium. Take a portion of the hyphae of the selected strain Actinomucor elegans CGMCC3.3393 and place it into the medium in the Erlenmeyer flask. Seal the mouth of the Erlenmeyer flask with gauze and wrap it. Incubate in a constant temperature shaker at 25℃ and 160rpm for 24h for later use.

[0020] Scale-up culture: Autoclaved PDB medium was poured into 1L Erlenmeyer flasks, each containing 400mL of medium, for a total of 60 flasks. Simultaneously, approximately 1mL of the above seed culture was added to each flask. The flasks were then sealed tightly with gauze and incubated at 25°C and 160rpm in a constant-temperature shaker for 24 hours. Once the strain reached the logarithmic growth phase, 20mg of arbutin (dissolved in anhydrous ethanol, approximately 2mL) was added to each flask, and the flasks were incubated for another week in a constant-temperature shaker.

[0021] 2) Extraction, separation and purification

[0022] After culturing for 7 days, the bacterial solution was filtered through four layers of non-woven gauze, and extracted three times with an equal amount of ethyl acetate. The ethyl acetate layer was collected, evaporated and concentrated to obtain 2.14g of crude product.

[0023] The crude product was dissolved in methanol and coarsely filtered. Initial separation was performed on an ODS-C18 open column (60cm × 3cm column, 50μm × 100g ODS-C18 column). The initial separation used a gradient elution with methanol:water (10:90, 40:60, 60:40, 80:20, 90:10, 100:0) as the mobile phase. The eluted components were combined by thin-layer chromatography and analytical high-performance liquid chromatography (HPLC), yielding four fractions (AD). Each fraction was further separated and prepared using semi-preparative HPLC.

[0024] Fraction B (127.7 mg) was prepared using acetonitrile:water (65:35, flow rate 3.0 mL / min) as the mobile phase to obtain a compound with the structural formula III.

[0025] Fraction D (174.2 mg) was prepared using acetonitrile:water (35:65, flow rate 3.0 mL / min) as the mobile phase to obtain compounds with structural formulas I, II, IV-IX.

[0026] The high-resolution mass spectrometry and NMR data of compounds I-IX are shown below.

[0027] Compound I: 3β,7β,15α,21β-tetrahydroxy-13β,28β-epoxy-10-double bond-ursane; optical rotation +31.5° (c 0.11, MeOH); High-resolution mass spectrometry m / z 533.3495 [M + COOH] - (ca l cd C 31 H 49 O7,533.3484); the proton and carbon NMR spectra are shown in Table 1.

[0028] Compound II: 3β,15α,21β-trihydroxy-13β,28β-epoxy-10-double bond-ursane; optical rotation +26.8° (c 0.12, MeOH); High-resolution mass spectrometry m / z 517.3526 [M + COOH] - (ca l cd C 31 H 49 O6,517.3535); the proton and carbon NMR spectra are shown in Table 1.

[0029] Compound III: 3β,7β-dihydroxy-21β-acetoxy-13β,28β-epoxy-10-double bond-ursane; optical rotation +33.1° (c 0.12, MeOH); High-resolution mass spectrometry m / z 537.3555 [M+Na] + (ca l cd C 32 H 50 O5Na, 537.3556); the proton and carbon NMR spectra are shown in Table 1.

[0030] Compound IV: 7β,15α,21β-trihydroxy-3β-acetoxy-13β,28β-epoxy-10-double bond-ursane; optical rotation +20.2° (c 0.11, MeOH); High-resolution mass spectrometry m / z 553.3498 [M+Na] + (ca l cd 553.3505,C 32 H 50 (O6Na); the 1H and 1C NMR spectra are shown in Table 2.

[0031] Compound V: 7β,21β-dihydroxy-3-carbonyl-13β,28β-epoxy-10-double bond-ursane; optical rotation +15.6° (c 0.14, MeOH); High-resolution mass spectrometry m / z 515.3370 [M + COOH] - (ca l cd C 31 H 47O6,515.3378); the proton and carbon NMR spectra are shown in Table 2.

[0032] Compound VI: 3β,7β,21β-trihydroxy-28-methoxy-13β,28β-epoxy-10-double bond-ursane; optical rotation +35.4° (c 0.12, MeOH); High-resolution mass spectrometry m / z 503.3732 [M+H] + (ca l cd C 31 H 51 O5,503.3731); the proton and carbon NMR spectra are shown in Table 2.

[0033] Compound VII: 3β,7β,15α,21β,23-pentahydroxy-13β,28β-epoxy-10-double bond-ursane; optical rotation +38.7° (c 0.11, MeOH); High-resolution mass spectrometry m / z 527.3354 [M+Na] + (ca l cd C 30 H 48 O6Na, 527.3349); the proton and carbon NMR spectra are shown in Table 3.

[0034] Compound VIII: 3β,7β,21β,24-tetrahydroxy-13β,28β-epoxy-10-double bond-ursane; optical rotation +48.2° (c 0.12, MeOH); High-resolution mass spectrometry m / z 533.3472 [M+COOH] - (ca l cd C 31 H 49 O7,533.3484); the proton and carbon NMR spectra are shown in Table 3.

[0035] Compound IX: 3β,7β,24-trihydroxy-21-carbonyl-13β,28β-epoxy-10-double bond-ursane; optical rotation +43.6° (c 0.13, MeOH); High-resolution mass spectrometry m / z 531.3322 [M+COOH] - (ca l cd C 31 H 47 O7,5331.3327); the proton and carbon NMR spectra are shown in Table 3.

[0036] Table 1. 1H and 1C NMR spectra of compounds I through III

[0037]

[0038]

[0039] Table 2. 1H and 1C NMR spectra of compounds IV through VI

[0040]

[0041]

[0042] Table 3. 1H and 1C NMR spectra of compounds VII-IX

[0043]

[0044]

[0045] The above results indicate that the structures of the obtained compounds, formulas I to IX, are correct.

[0046] Using the microbial transformation method described above, novel ursane-type triterpenoids with structural formulas I to IX were also obtained by transforming strains of *Mucor erectus* CGMCC 3.180 and *Mucor racemosus* CGMCC 3.20, respectively.

[0047] Example 2: Antitumor activity of novel ursane-type triterpenes with structural formulas I-IX

[0048] 1) Experimental methods

[0049] The half-maximal inhibitory rate (IC50) of each compound against tumor cell lines was determined using the MTT assay. 50 Value. The experimental method is as follows:

[0050] Tumor cells in the logarithmic growth phase were collected and their concentration adjusted to 5 × 10⁶ cells / mL using RPM I 1640 culture medium containing 10% fetal bovine serum. 5 Cells were seeded at 100 μL / well in 96-well culture plates. The experiment included a blank control group, a cell control group, and a drug treatment group. The cell control group and drug treatment group received 100 μL of cell suspension per well, while the blank control group received only 100 μL of culture medium per well. After pre-culturing tumor cells in an incubator for 24 h, different concentrations of the test drug were added to achieve a final concentration of 0.1–100 μM, and the cells were cultured for another 48 h. The absorbance (A) at 570 nm was measured using the MTT assay, and the inhibition rate was calculated [Inhibition rate = (1 - A value of experimental group / A value of control group) × 100%]. The experiment was repeated three times. SPSS 11.5 software was used to perform regression equations to calculate the half-maximal inhibitory concentration (IC50) of each test sample on tumor cells after 48 h of treatment. 50 ).

[0051] 2) Experimental Results

[0052] Based on the MTT assay results, the IC50 of the test sample for the above-mentioned cells was calculated. 50 The values ​​are shown in Table 4.

[0053] Table 4. Screening results of in vitro cytotoxic activity of test samples

[0054]

[0055] The results show that the compounds with structural formulas I to IX of this invention have good antitumor activity and can be used as active ingredients in antitumor drugs.

Claims

1. A 13,28-epoxy-substituted ursane-type triterpenoid derivative or a pharmaceutically acceptable salt thereof, characterized in that, The structural formula of the 13,28-epoxy-substituted oleanane type triterpene derivative is selected from any one of the following structural formulae:

2. A process for the microbial conversion of a 13, 28-epoxy-substituted us- san-type triterpene derivative according to claim 1, characterized in that, The microbial transformation preparation method is as follows: taking ursolic acid as a substrate, using Actinomucor elegans CGMCC 3.3393 to perform microbial transformation, and obtaining the 13,28-epoxy-substituted oleanane type triterpene derivative.

3. Application of the 13,28-epoxy-substituted oleanane type triterpene derivative in claim 1 in preparation of an anti-tumor drug, wherein the tumor is selected from one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

4. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The active ingredient of the anti-tumor drug is the 13,28-epoxy-substituted oleanane type triterpene derivative in claim 1, and the tumor is selected from one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

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