阅读说明：本技术 一种紫罗兰酮生物碱衍生物及医药用途 (Ionone alkaloid derivative and medical application thereof ) 是由 段宏泉 秦楠 陈莹 聂江平 吴潇然 樊晔 于 2020-03-19 设计创作，主要内容包括：本发明提供一种具有抗乳腺癌转移作用的紫罗兰酮生物碱衍生物或其药学上可接受的盐,其结构如式I：<Image he="780" wi="700" file="DSA0000204291450000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>本发明还提供了该类紫罗兰酮生物碱衍生物在制备抗乳腺癌转移药物方面的用途。(The invention provides an ionone alkaloid derivative with an anti-breast cancer metastasis effect or a pharmaceutically acceptable salt thereof, and the structure of the derivative is shown as the formulaI： The invention also provides application of the ionone alkaloid derivative in preparing a medicine for resisting breast cancer metastasis.)

1. An ionone alkaloid derivative or a pharmaceutically acceptable salt thereof, the structure of which is shown in the general formula I:

2. the ionone alkaloid derivative according to claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of compounds N2, 7, 8, 10, 18, 26, 43.

3. An ionone alkaloid derivative according to claim 2, or a pharmaceutically acceptable salt thereof, selected from compound N8, 18, 43.

4. Use of the ionone alkaloid derivative according to any of the claims 1-3 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament against breast cancer metastasis.

Technical Field

The invention relates to an ionone alkaloid derivative, a preparation method and application thereof as a medicine for resisting breast cancer metastasis, belonging to the field of pharmaceutical chemistry research.

Background

According to the report of "2018 global cancer statistics" published in journal of the american cancer society, "journal of clinicians cancer", there will be approximately 1810 ten thousand new cases of cancer and 960 ten thousand cases of cancer death globally in 2018. Of the 1810 new cancer cases, asia accounts for nearly half, and of the 960 ten cancer dead patients, asia accounts for nearly seven months, and by 2030, the number of new tumor cases will increase by about 50%. Therefore, tumors have become one of the major diseases that are currently seriously threatening human life and health. In a large number of clinical cases, tumor metastasis is the leading cause of death in malignant patients, and the cause of death in about 90% of malignant patients is associated with tumor metastasis. Therefore, the basic mechanism of tumor metastasis is deeply understood, a new strategy for inhibiting tumor metastasis is sought, and the discovery of an anti-tumor metastasis drug is the key to effectively preventing and treating malignant tumor metastasis.

Tumor metastasis is one of the important biological characteristics of malignant tumors, and invasion and metastasis of tumor cells are processes of multiple stages, multiple steps, multiple factors and multiple gene-coordinated interactions. Metastasis of tumor cells is generally considered to include the following four stages: (1) the adhesion capacity among tumor cells is reduced, and the tumor cells are separated from a primary tumor focus and adhered to a basement membrane; (2) the fallen tumor cells are autocrine and induce the tumor interstitial cells to secrete a plurality of proteases to degrade extracellular matrix and basement membrane and migrate to invade blood vessels; (3) invasion of the circulatory system, migration and adhesion of vascular endothelial cells to new sites with blood; (4) after penetrating the vessel wall, tumor cells colonize, proliferate, and eventually form metastases in specific tissues or organs.

For example, Cilengitide, a phase III clinical drug that inhibits tumor cell adhesion using integrin as a target, acts on α v β 3 receptor and α v β 5 receptor, and inhibits angiogenesis against glial cells^[4]. Acting on matrix metalloproteinsThe enzyme (MMP) medicine Batimastat (BB-94) can obviously inhibit the activities of various MMP subtypes, further inhibit the invasion of tumor cells by using MMP, and inhibit angiogenesis. Vascular Endothelial Growth Factor (VEGF) is a central regulator of neovascularization, and can increase the permeability of blood vessels and provide favorable conditions for the metastasis and diffusion of tumor cells. Vandetanib (ZD-6474) inhibits tumor angiogenesis by acting on VEGF receptors, and also inhibits tumor cell proliferation.

Thus, interfering with or blocking the process of malignant tumor dissemination in the host is the most effective means to prolong survival, improve quality of life and reduce mortality. On the other hand, because the conventional anti-tumor metastasis drug treatment after primary tumor resection is a continuous and long-term administration process, the ideal anti-tumor metastasis drug not only blocks the invasion and metastasis of tumor cells in vivo, but also is a non-cytotoxic drug or has low toxic and side effects, and cannot damage the immune system. Although the tumor chemotherapy drugs can kill or inhibit the growth of tumors, the tumor chemotherapy drugs are cytotoxic drugs, so that the tumor chemotherapy drugs can kill tumor cells and cause great damage to normal cells. Therefore, the ideal anti-tumor metastasis medicine should be a non-cytotoxic medicine with low toxic and side effects.

116) The invention patent of the research group discloses a common clubmoss herb alkaloid compound (Shen 201010114809.9) with the function of resisting tumor metastasis, further takes an active ingredient Salignone as a lead to synthesize a series of derivatives, and discloses an invention patent pregnane alkaloid derivative with the function of resisting breast cancer metastasis and medical application (patent number 201310749522.7). . The research group carries out derivative Synthesis on ionone alkaloids in the grass of Trans-tinospora, and the found anti-tumor metastasis lead compound ION-31a [ Synthesis and anti-metastatic effects of novel chiralionone alkaloid derivatives, Hai-Jun Fang, Xiao-Ai Shou, Qian Liu, Chun-Chun Gan, Hong-Quan Duan, Nan Qin, European Journal of Medicinal Chemistry 101(2015) 245-.

Disclosure of Invention

The invention aims to provide an ionone alkaloid derivative and application of the compound in preparing a medicine for resisting breast cancer metastasis.

The invention is realized by the following technical scheme:

on one hand, the invention provides an ionone alkaloid derivative or a pharmaceutically acceptable salt thereof, and the structure and the synthetic route of the derivative are shown as formulas 1-4:

formula 1 synthesis route of ionone alkaloid derivative N1-N28

Synthesis route of ionone alkaloid derivative N29-N40 of formula 2

Synthesis route of ionone alkaloid derivative N41-N48 of formula 3

Synthesis route of ionone alkaloid derivative N49-N73 of formula 4

In another aspect, the invention provides a pregnane alkaloid derivative or a pharmaceutically acceptable salt thereof for use in preparing a medicament for resisting breast cancer metastasis.

First, the present invention has performed cytotoxicity assay on the synthesized pregnane alkaloid derivative N1-N73, and further performed anti-breast cancer metastasis activity screening in a non-cytotoxic dose.

Toxic effects of the respective derivatives on breast cancer cells

Principle of MTT colorimetric assay: this assay is based on the reduction of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) and is a commonly used method for determining cell survival and growth. MTT is a universal cell stain, and the enzyme disappears and MTT is not reduced when exogenous yellow MTT is reduced into insoluble blue-violet crystal formazan (formazan) by the existence of succinate dehydrogenase related to NADP in mitochondria of living cells and is deposited in the cells. After dissolving the Formazane in DMSO, the change of the optical density is detected at the wavelength of 550nm by an enzyme-labeling instrument to measure the growth inhibition effect of the test drug on the cells, and then the cytotoxicity is evaluated.

The experimental steps are as follows: digesting cultured cells with 0.25% pancreatin, sucking out pancreatin, stopping digestion with 10% FBS-containing culture medium, mixing cell suspension, and counting to adjust density to 1 × 10⁴Adding the prepared cell suspension to a 96-well plate, placing 180ul of the cell suspension per well at 37 ℃ and 5% CO₂Culturing in incubator for 24 hr, adding medicine after 24 hr, each concentration is 5 multiple holes, adding medicine, placing at 37 deg.C and 5% CO₂After 48 hours of incubation, 5mg/ml of MTT20ul was added to each well, the mixture was incubated at 37 ℃ for 4 hours in a 5% CO2 incubator, and after 4 hours the 96-well plate was removed, the supernatant was aspirated, 100ul of DMSO was added to each well, and the absorbance was measured at a wavelength of 570nm using a microplate reader. The inhibition rate is (1-addition medicine OD value/blank group OD value) x 100%

Principle of method for evaluating Transwell chemotaxis of breast cancer metastasis resistance activity

The size of the cell motility can be analyzed by using a model in which a filter membrane with an 8 μm pore size is directly placed between the upper and lower chambers of the invasive chamber in the upper chamber of the Transwell chamber, and tumor cells move through the filter membrane by deformation. When the drug is screened, a chemotaxis inducer (chemotactic factor EGF) is added, the change of the number of cells which are chemotactic and penetrate through a filter membrane by the tumor cells co-cultured with the test drug and the blank group of tumor cells is measured, so that the chemotactic migration capacity of the test drug for inhibiting the tumor cells is expressed, and the activity of the test drug for resisting tumor metastasis is evaluated. The positive control comprises a reagent LY294002 (a PI3K inhibitor with the function of resisting tumor metastasis) and a lead compound ION-31 a.

The experimental steps are as follows:

1. screening of non-cytotoxic amounts of sample: the MTT method is used to screen out the sample concentration without cytotoxic effect.

2. Co-culturing the sample with the cells: the cells were plated in 6-well plates at 37 ℃ with 5% CO₂Is cultured in an incubator for 24 hours to be attached to the wall, and after 24 hours, the sample is added into a 6-well plate at a determined concentration and 5% CO at 37 DEG C₂Incubate for 24 hours.

3. Chemotaxis experiment: after the cells treated with the sample were digested with 0.25% of trypsin, the digestion was terminated with a culture medium containing 10% FBS, and the cell suspension was centrifuged at 1300rpm for 5 minutes. The supernatant was decanted, 0.1% BM was added to mix the cells well, centrifuged again for 5 minutes, counted and the cell density adjusted to 5X10⁵And placing the mixture in an incubator for later use. Preparing chemotactic factor EGF on ice, adding to the lower chamber of chemotactic chamber, each well having 30ul, spreading the coated membrane on the lower chamber, placing the gel pad, fixing the upper chamber, adding the prepared cell suspension to the upper chamber, each well having 50ul and each sample having 3-fold wells, adding into CO₂Culturing in incubator for 3.5 hr, taking out, scraping cells without chemotaxis, fixing and staining with three-step staining reagent, fixing membrane with paraffin oil, and counting under microscope.

4. The statistical calculation method comprises the following steps: during counting, 5 fields of view are selected for each well to be counted respectively, the average value is taken, and the average value of the three multiple wells is taken again to obtain the number of chemotactic cells under the concentration of the sample. The data were processed with SPSS11.5 to calculate the median suppression ratio IC₅₀。

Calculating the formula:

results of the experiment

Half the Inhibitory Concentration (IC) of each preferred derivative on the chemotactic migration of MB-MDA-231 (human breast cancer cells)₅₀) As shown in table 1:

TABLE 1 half Inhibitory Concentration (IC) of each derivative inhibiting chemotactic migration of breast cancer cells₅₀)

Evaluation of anti-metastatic activity against breast cancer shows that the preferred compounds all show anti-metastatic activity against breast cancer at non-cytotoxic amounts, with the activity of compounds N2, 5, 7, 8, 10, 13, 15, 18, 19, 21, 23-27, 29, 37-39, 43, 58-61, 66, 71 being particularly prominent.

Detailed Description