CN113134006B - Application of ursolic acid derivative in preparing antitumor drugs - Google Patents

Abstract

The invention belongs to the field of medicines, and discloses an ursolic acid derivative or a pharmaceutically acceptable salt thereof and application thereof in preparing an anti-tumor medicine. The invention successfully carries out structural modification on ursolic acid by utilizing a microbial transformation technology to obtain 4 novel ursolic acid derivatives with mother nucleus structural modification, and in-vitro anti-tumor cell tests prove that the compounds have better anti-tumor activity, can be used as active ingredients of anti-tumor drugs and have wide application.

Description

Application of ursolic acid derivative in preparing antitumor drugs

Technical Field

The invention relates to the field of medicines, in particular to application of ursolic acid derivatives in preparation of antitumor drugs.

Background

Ursolic acid, also known as ursolic acid, is a pentacyclic triterpene with a special structure and exists in various Chinese herbal medicines such as dogwood, glossy privet fruit, Chinese magnoliavine fruit and the like. Modern pharmacological research finds that ursolic acid has the effects of resisting tumors, resisting oxidation, resisting inflammation, reducing blood sugar, resisting hepatic fibrosis and the like. In addition, there have been many studies on the neuroprotective effect of ursolic acid in recent years. In the aspect of chemical structure modification, the research on the anti-tumor aspect of the chemically synthesized derivatives of ursolic acid is abundant, but the sites of chemical structure modification are mainly limited to derivatization of the hydroxyl at the 3-position and the carboxyl at the 28-position of ursolic acid. Due to the structural particularity of the pentacyclic triterpenoid, the parent nucleus lacks active groups, the chemical reaction sites are few, and the derivative with hydroxyl, carbonyl and other modifications on the parent nucleus is difficult to obtain by adopting a conventional chemical reaction method. Therefore, the chemical and pharmacological research of the ursolic acid derivative with the mother nucleus structure modification is not comprehensive.

Microbial transformation is an enzyme-catalyzed reaction carried out by utilizing an enzyme system with the self-specificity of an organism, has multiple reaction types and high stereoselectivity and regioselectivity, and becomes an important tool in organic synthesis. The application of the derivative in the preparation of the ursolic acid can provide a large number of samples for the subsequent research of the compounds.

Disclosure of Invention

In view of the above, the present invention provides an application of ursolic acid derivatives in the preparation of anti-tumor drugs, and particularly provides an application of ursolic acid derivatives or pharmaceutically acceptable salts thereof in the preparation of anti-tumor drugs, and the ursolic acid derivatives have good anti-tumor activity. In the invention, the ursolic acid derivative is a compound with the structural formula of formula I-formula IV:

the invention also provides a preparation method of the ursolic acid derivative, which comprises the following steps:

1) fermenting and culturing microorganisms, adding ursolic acid into a culture medium, then performing transformation culture, and removing mycelia to obtain a fermentation liquid, wherein the microorganisms are strains of the genus Torulopsis (Circinella); preferably, the strain is Byssochlamus terricola moscae CGMCC 3.2695.

2) Extracting the fermentation liquor, and evaporating the extract to obtain a converted crude extract;

3) and purifying the transformed crude extract by using reverse phase high performance liquid chromatography to obtain a product. Wherein, the preparation conditions of the reversed-phase high performance liquid chromatography are that A semi-preparative chromatographic column YMC ODS-A is 10.0 I.D. times.250 mm, acetonitrile-water (45:55, V/V), the flow rate is 2.5mL/min, and the detection wavelength is 203 nm.

The concentration of the ursolic acid in the culture medium in the step 1) of the method is 2-5000 mug/mL.

The extraction solvent in the step 2) of the method is a conventional organic solvent, preferably ethyl acetate.

Experiments prove that the ursolic acid derivative has good anti-tumor activity and can be used as an active ingredient of an anti-tumor medicament. The active ingredients of the antitumor drugs can be one or more compounds with structural formulas of formula I, formula II, formula III and formula IV.

In the medicine taking the compound as the active ingredient, one or more pharmaceutically acceptable carriers can be added when necessary. The carrier comprises a diluent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant and the like which are conventional in the pharmaceutical field, and can be prepared according to a conventional method in the pharmaceutical field.

The invention successfully carries out structural modification on the ursolic acid by utilizing a microbial transformation technology to obtain a new ursolic acid derivative, and the compounds have better anti-tumor activity and can be used as active ingredients of anti-tumor medicaments and have wide application as proved by in vivo animal experiments and in vitro anti-tumor cell experiments.

Drawings

FIG. 1 is an HPLC liquid chromatogram of the ursolic acid derivative of the present invention.

Detailed Description

Example 1 preparation of Compounds of formulae I-IV

The compound is prepared by adopting a microbial conversion method and using ursolic acid as a raw material through the steps of fermentation, extraction, separation and the like. The strain of Torulopsis (Circinella) can be purchased from China academy of sciences microorganism culture Collection management center (CGMCC), and is preserved in a solid slant culture medium at 4 deg.C in a refrigerator.

Taking Circinella muscsae CGMCC 3.2695 as an example, the process for preparing the compound with the structural formula I-VI is as follows:

1) fermentation, transformation and extraction

The Circinella muscae CGMCC 3.2695 was inoculated into 2 250mL triangular flasks (containing 100mL potato medium) as seed solutions. After shaking culture on a shaking table at 160rpm and 26 ℃ for 1 day, 1mL of seed solution was aspirated by a sterile pipette and added to 20 1000mL shake flasks (containing 400mL potato medium) until the hyphae growth was in vigorous phase. After 1 day of shake culture, 25mg of ursolic acid (0.2mL, 125mg/mLDMSO solution) was added to each flask, sharing 500mg of substrate. Continuing to transform for 5 days under the same conditions, filtering the fermentation liquor, filtering to remove mycelium, extracting the filtrate with equal volume of ethyl acetate for 3 times, and concentrating the extractive solution under reduced pressure to dryness to obtain about 0.83g of crude extract of the transformed matter.

2) Purification by reversed phase high performance liquid chromatography

The combined fractions were purified by reverse phase high performance liquid chromatography. The preparation conditions were a semi-preparative column YMC ODS A-5 μm, 10.0X 250mm, acetonitrile-water (45:55, V/V), flow rate 2.5mL/min, and detection wavelength 203 nm. Obtaining 4 transformation products of the compounds with the structural formulas I-IV, wherein the chromatogram is shown in figure 1¹³The C-NMR data are shown in Table 1.

TABLE 1 carbon spectra data (deuterated pyridines) of Compound I, Compound II, Compound III and Compound IV

The above results indicate that the obtained compound has the correct structure.

EXAMPLE 2 antitumor Activity of Compound I, Compound II, Compound III and Compound IV of the present invention

1) Experimental Material

Instruments and reagents: CO 2₂Incubator (Joean IGO 150); microplate reader (Bio-TEK ELx 800); fluorescence inverted microscope (Olympus IX 51); MTT cell proliferation and cytotoxicity detection kit (Biyuntian biotechnological research institute), RPM I1640 culture medium (Gibcol BRL), RNase A, fetal bovine serum, dimethyl sulfoxide (DMSO), and trypsin (Shanghai bioengineering Co., Ltd.).

Tumor cell lines were tested: 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), and CT26 cells (colon cancer cells), which were purchased from the institute of tumor research of the Chinese academy of medicine and sciences.

Test samples: the purity of the ursolic acid and the compounds I-IV synthesized in the embodiment 1 is more than 95 percent; meanwhile, cisplatin is selected as a positive control drug, and each compound is dissolved in DMSO and then diluted.

2) Experimental methods

Determination of half inhibition rate IC of each tested compound on tumor cell strain by MTT method₅₀The value: taking tumor cells in logarithmic growth phase, adjusting cell concentration to 5 × 10 with RPM I1640 culture solution containing 10% calf serum⁵and/mL, inoculating the cells in a 96-well culture plate, adding 100 mu L of cell suspension into each well of a drug treatment group and a cell control group, setting 3 multiple wells in each group, adding only RPM I1640 full culture medium into a blank control group, setting 100 mu L of cell suspension into each well, and setting 3 multiple wells. Placing 96-well culture plate at 37 deg.C and 5% CO₂After 24h of incubation in an incubator, test samples of different concentrations were added to a final concentration of 0.1-100. mu.M, and incubation was continued for 72 h. Measuring absorbance (A) value at 570nm by MTT method in microplate reader, and calculating inhibition rate (1-experiment group A value/control group A value) × 100%]. The experiment was repeated 3 times. Using SPSS 11.5 software as regression equation, calculate half maximal Inhibitory Concentration (IC) of each test sample for 72h on tumor cells₅₀)。

3) Results of the experiment

According to the MTT method test result, calculating the IC of the ursolic acid and the compounds I-IV of the invention on the cells₅₀The results are shown in Table 2.

TABLE 2 in vitro cytotoxic Activity screening results of test samples

The results show that the compounds I-IV have good antitumor activity and can be used as active ingredients of antitumor drugs.

It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (2)

1. The application of ursolic acid derivative or its pharmaceutically acceptable salt with any one of the following structural formulas in preparing antitumor drugs,

the tumor is cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer or colon cancer.

2. The use as claimed in claim 1, wherein the antitumor drug comprises an active ingredient and a pharmaceutically acceptable carrier, the active ingredient is selected from one or more of ursolic acid derivatives of formula I, formula II, formula III and formula IV or pharmaceutically acceptable salts thereof,

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