CN112142819A

CN112142819A - Application of betulinic acid derivative in preparation of antitumor drugs

Info

Publication number: CN112142819A
Application number: CN202010959717.4A
Authority: CN
Inventors: 陈广通; 宋开南; 张永珍; 储呈娇; 宋妍; 陈婷婷
Original assignee: Nantong University
Current assignee: Shanghai kepeptide Biotechnology Co.,Ltd.
Priority date: 2020-09-14
Filing date: 2020-09-14
Publication date: 2020-12-29
Anticipated expiration: 2040-09-14
Also published as: CN112142819B

Abstract

The invention belongs to the field of medicines, and discloses a betulinic 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 the betulinic acid by utilizing a microbial transformation technology to obtain a plurality of novel compounds, 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 betulinic acid derivative in preparation of antitumor drugs

Technical Field

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

Background

Betulinic acid is a kind of pentacyclic triterpenoid with special structure. The structure is characterized in that the E ring is a five-membered carbon ring, isopropyl at the 19-position of the E ring is substituted by alpha-configuration, and five rings are arranged in a trans-form. The ingredients are mainly distributed in birch bark, spina date seed, radix asparagi, Chinese pulsatilla root and other traditional Chinese medicines and are main effective ingredients of the traditional Chinese medicines. Modern pharmacological research finds that the betulinic acid has a remarkable anti-tumor effect and has an obvious inhibiting effect on various tumor cells of lung cancer, ovarian cancer, cervical cancer, breast cancer, melanoma and the like. Unlike traditional chemotherapeutic drugs, which typically kill normal cells, betulinic acid has selective human tumor cytotoxicity, which makes it one of the most potent anti-tumor compounds.

Semisynthetic derivatives of natural compounds play an important role in the research of new drugs, and the pharmacological activity of the compounds can be obviously changed by structural modification. However, pentacyclic triterpenoid has special structure, lack of active groups in parent nucleus and few reaction sites, and the derivatives meeting the requirements are difficult to prepare by adopting a conventional chemical reaction method.

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 betulinic acid derivative in preparation of the betulinic acid derivative can provide a large number of samples for subsequent research of the compounds.

Disclosure of Invention

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

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

1) fermenting and culturing microorganisms, adding betulinic acid into a culture medium, then performing transformation culture, and removing mycelium to obtain a fermentation broth, 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 (30:70, V/V), the flow rate is 2.5mL/min, and the detection wavelength is 203 nm.

The concentration of betulinic 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 betulinic 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 selected from the compounds with structural formulas of formula I, formula II, formula III, formula IV, formula V and formula VI.

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 betulinic acid by utilizing a microbial transformation technology to obtain a new betulinic acid derivative, and the compounds have better antitumor activity and can be used as active ingredients of antitumor drugs and have wide application as proved by in vivo animal experiments and in vitro antitumor cell experiments.

Drawings

FIG. 1 is an HPLC liquid chromatogram of betulinic acid derivatives according to the present invention.

Detailed Description

Example 1 preparation of Compounds of formula I-VI

The compound is prepared by adopting a microbial conversion method and taking betulinic 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, 20mg betulinic acid (0.2mL, 100mg/mLDMSO solution) was added to each flask, sharing 400mg substrate. Continuing to transform for 7 days under the same conditions, filtering the fermentation broth, 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 0.56g 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 (30:70, V/V), flow rate 2.5mL/min, and detection wavelength 203 nm. Obtaining 6 transformation products of the compound with the structural formula I-VI, wherein the chromatogram is shown in figure 1¹³The C-NMR data are shown in Table 1.

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

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

EXAMPLE 2 antitumor Activity of Compound I, Compound II, Compound III, Compound IV, Compound V and Compound VI of the 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 betulinic acid and the compound I-VI synthesized in the example 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 serum⁵/mL, inoculated in 96-well culture plates, drug-treated group and cell control group added to each well100 mu L of cell suspension, each group is provided with 3 multiple wells, the blank control group is only added with RPM I1640 full culture medium, each well is 100 mu L, and 3 multiple wells are arranged. 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 IC of betulinic acid and the compounds I-VI 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-VI 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

1. The application of betulinic acid derivative or its pharmaceutically acceptable salt in preparing antitumor medicine,

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

3. the use of claim 1 or 2, wherein the tumor is cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer or colon cancer.