CN114010622B - Application of fluorine-containing compound in preparation of medicines for treating cancers - Google Patents

Abstract

The invention belongs to the field of cancer treatment, and particularly relates to application of fluorine-containing compounds in cancer treatment. The present invention provides a medicament for treating cancer, and in particular, a medicament for treating lung cancer selected from bromobenzene substituted trifluoromethyl benzocyclopentanone, to extend the life of cancer patients. The invention shows that bromobenzene substituted trifluoromethyl benzocyclopentanone inhibits the proliferation of lung cancer A549 cells according to concentration tolerance, and the result of the invention reveals that bromobenzene substituted trifluoromethyl benzocyclopentanone has the activity of resisting lung cancer proliferation and possible action mechanism thereof, thereby providing a potential target molecule for the research and development of cancer chemotherapeutic drugs.

Description

Application of fluorine-containing compound in preparation of medicines for treating cancers

Technical Field

The invention belongs to the field of cancer treatment, and particularly relates to application of bromobenzene substituted trifluoromethyl benzocyclopentanone in cancer treatment.

Background

Cancer is a disease caused by the loss of normal regulation and hyperproliferation of body cells. Hyperproliferative cells are called cancer cells, which can often invade surrounding tissues and can even metastasize to other parts of the body via the circulatory system and/or lymphatic system (cancer metastasis).

Meanwhile, lung cancer has become the first cancer of the mortality rate, the number of new diseases per year is still high, and lung cancer has become one of diseases that seriously affect human health (Siegel RL, miller KD, jemal A: cancer statistics,2020.CA Cancer J Clin 2020,70 (1): 7-30). Although early diagnosis and treatment of lung cancer have been greatly improved with the development of medical technology, patients still have a survival rate of less than 15% for 5 years (Yan L, zhang Y, li K, wang M, li J, qi Z, wu J, wang Z, ling L, liu H et al: miR-593-5p inhibit cell proliferation by targeting PLK1 in non small cell lung cancer cells.Pathol Res Pract 2020,216 (2): 152786).

Thus, research and discovery of new therapeutic agents for treating cancer, especially for lung cancer, will play a very important role in improving the quality of life of patients.

Disclosure of Invention

(one) solving the technical problems

The invention provides a medicine for treating cancers, in particular to a medicine for treating lung cancer, so as to prolong the service life of cancer patients.

(II) technical scheme

To achieve the above object, in one embodiment, the present invention provides the use of a compound i for the preparation of a medicament for the treatment of cancer, said compound i having the structural formula:

preferably, the cancer is selected from lung cancer, liver cancer, stomach cancer, esophageal cancer, colorectal cancer, leukemia, brain tumor, nasopharyngeal cancer, pancreatic cancer, bladder cancer, breast cancer, bone cancer, cervical cancer.

Preferably, the cancer is selected from lung cancer.

In one embodiment, the present invention provides a pharmaceutical formulation comprising compound i having the formula:

preferably, the formulation is selected from a liquid dosage form, a solid dosage form, a semi-solid dosage form or a gaseous dosage form.

Preferably, the liquid dosage form comprises a solution, an injection, an infusion or an oral liquid.

Preferably, the solid dosage form comprises a tablet, capsule, powder or granule.

Preferably, the semi-solid dosage form comprises an ointment or gel.

Preferably, the gaseous dosage form comprises an aerosol or a spray.

In another embodiment, the invention provides the use of any of the above pharmaceutical formulations for the preparation of a medicament for the treatment of cancer; preferably, the cancer is selected from lung cancer, liver cancer, stomach cancer, esophageal cancer, colorectal cancer, leukemia, brain tumor, nasopharyngeal cancer, pancreatic cancer, bladder cancer, breast cancer, bone cancer, cervical cancer; preferably, the cancer is selected from lung cancer.

(III) beneficial effects

The compound I provided by the invention inhibits the proliferation of lung cancer A549 cells in a concentration-dependent manner, and the result of the invention reveals that the compound I has the activity of resisting lung cancer proliferation and a possible action mechanism thereof, thereby providing a potential target molecule for the research and development of lung cancer chemotherapeutic drugs.

Drawings

The structural formula of the compound I in FIG. 1

FIG. 2 measurement of the extent of influence of Compound I on A549 cell viability by CCK-8 method

FIG. 3 detection of the extent of influence of Compound II on A549 cell viability using CCK-8 method

FIG. 4 measurement of the extent of influence of Compound III on A549 cell viability by CCK-8 method

FIG. 5 detection of the extent of influence of Compound IV on A549 cell viability by CCK-8 method

FIG. 6 influence of Compound I on proliferation of A549 cells

FIG. 7 Western blot method for detecting influence of Compound I on cell proliferation-related Signal pathway

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The following is a detailed description of specific examples.

The human lung cancer cell strain A549 is purchased from Shanghai cell bank of China academy of sciences, fetal bovine serum is purchased from Sigma, and EdU kit is purchased from Shangbo biotechnology Co., ltd; CCK-8 kit was purchased from Beijing Kaiki Biotech Co., ltd; p-AKT, AKT, p-mTOR, and GAPDH primary antibodies were purchased from Cell Signaling Technology; the secondary antibody was purchased from Santa Cruz.

The structural formula of the compound I-compound IV is as follows:

compound I (code XX 0235)

Compound II (code XX 00125)

Compound III (code XX 00126)

Compound IV (code XX 00136)

Data processing and statistical analysis employed SPSS19.0 software. All data are expressed as mean ± standard deviation (mean ± SD), the comparison between groups uses one-way analysis of variance, and the difference is statistically significant as P < 0.05.

EXAMPLE 1 Compound I-Compound IV inhibiting A549 cell viability

Lung cancer a549 cells were cultured at 37 ℃ with 5% CO ₂ In the environment, the culture solution formula is DMEM complete medium containing 10% of fetal calf serum. After the cells were cultured to log phase, subsequent experiments were performed.

After treating A549 cells with fluorochemicals-Compound I-Compound IV at concentrations of 6.25, 12.5, 25 and 50. Mu.g/mL for 24h, cell viability was measured by CCK-8 method. The results show that: concentration of 6 compared with DMSO control groupThe relative viability of the cells after treatment with compound i (XX 0235) at 25, 12.5, 25 and 50 μg/mL decreased, respectively, and exhibited decreased concentrations, indicating that compound i can inhibit the viability of a549 cells in dose-dependent fashion (see figure 2). IC of Compound I was calculated using Graphpad software (V8.0) ₅₀ The value was 41.25. Mu.g/mL (FIG. 2). The relative viability of a549 cells did not show a decrease in concentration after compound ii-compound iv treatment, and there was no effect of dose-dependent tolerance on inhibiting the viability of a549 cells (see figures 3-5).

The CCK-8 method for detecting the cell viability specifically comprises the following steps: human lung cancer cell A549 was packed at a density of 5X 10 per well ³ Individual cells, seeded into 96-well plates. After cell attachment, treated cells were added at concentrations (6.25, 12.5, 25 and 50. Mu.g/mL). After 24 hours, CCK-8 reagent was added to a concentration of 2.5g/L per well, and the culture was continued for 2 hours, and the absorbance (A) value per well was measured at a wavelength of 450nm using an enzyme-labeled instrument. The calculation formula of the relative activity of the cells is as follows: cell relative viability = experimental group _{A450 value} Blank group _{A450 value} 。

EXAMPLE 2 Compound I inhibits proliferation of A549 cells

The effect of compound i on a549 cell proliferation was tested using the EdU kit. The experimental results showed that relative numbers of EdU positive cells after treatment with compound i at concentrations of 6.25, 12.5, 25 and 50 μg/mL decreased by 29% (P < 0.05), 42% (P < 0.01), 50% (P < 0.01) and 61% (P < 0.0001), respectively, compared to DMSO control, as shown in fig. 6, demonstrating that compound i can inhibit proliferation of a549 cells in a concentration-dependent manner.

The EdU kit is used for detecting the proliferation of cells and comprises the following specific steps: human lung cancer cell A549 was packed at a density of 3X 10 per well ⁵ Individual cells, seeded into 24-well plates. Following cell attachment, cells were treated with compound I at concentrations (0, 6.25, 12.5, 25 and 50. Mu.g/mL). After 24h, the EDU solution (reagent A) was diluted with cell complete medium at a ratio of 1000:1 to prepare 300. Mu.L of 50. Mu.M EdU medium, which was incubated for 2h at 37 ℃; washing the cells with PBS 3 times for 5min each; then 150. Mu.L of 4% paraformaldehyde was added to each well and the mixture was fixed for 30min; then the fixed liquid is discarded, 150 mu L glycine with the concentration of 2mg/mL is added into each hole, and the mixture is incubated for 5min;discarding the solution, adding PBS to decolorize and shake the cells for 3 times, and 5min each time; adding 300 μl of penetrant (PBS containing 0.5% TritonX 100) into each well, decolorizing with shaking table, incubating for 10min, and washing with PBS for 1 time; then adding 300 mu L of Apollo staining solution into each hole, and incubating for 30min by a shake table at room temperature in dark place; washing with penetrant for 2-3 times for 10min each time; finally, preparing a proper amount of 1 Xhoechst 33342 reaction solution for DNA staining, adding 300 mu L of staining solution into each hole, incubating for 30min by a light-shielding, room temperature and decolorizing shaking table, and then washing for 3 times by PBS; immediately after the completion of staining, observation was performed by a fluorescence inverted microscope and photographing was performed.

EXAMPLE 3 Compound I inhibits proliferation of A549 cells by inhibiting Akt-mTOR signaling pathway

CCK-8 and EdU experiments show that the compound I inhibits the activity and proliferation of lung cancer A549 cells, so that a Western blot method is adopted to measure signal paths related to cell proliferation. The results show that: in comparison to the DMSO control, the levels of AKT and mTOR phosphorylation were significantly inhibited after treatment of cells with compound i at concentrations of 6.25, 12.5, 25 and 50 μg/mL, without any change in the background protein expression levels (see fig. 7).

The Western blot method is used for measuring a cell proliferation related signal path so as to detect the cell proliferation related protein expression, and specifically comprises the following steps: human lung cancer cell A549 was packed at a density of 6X 10 per well ⁵ Individual cells, seeded into 12-well plates. Following cell attachment, cells were treated with compound I at concentrations (6.25, 12.5, 25 and 50. Mu.g/mL). After 24h, the cells were collected and lysed by RAPI addition, and the supernatant was collected by centrifugation at 12000 Xg for 5min. The BCA method determines protein concentration of each group of samples and adds 5 x protein loading buffer, 100 ℃, for 5min. SDS-PAGE was performed according to the protein concentration of each group in an amount of 80. Mu.g of protein, and after completion of the electrophoresis, the proteins were transferred to PVDF membrane. PVDF membranes were blocked with 5% BSA in TBST and incubated for 1h at room temperature. Diluted primary antibody (1:1500) was then added and incubated overnight at 4 ℃. TBST was washed 3 times for 10min each. Diluted secondary antibodies (1:5000) were added and incubated for 1h at room temperature, and TBST was washed 3 times for 10min each. The cleaned film is put into an exposure cassette, the right side faces upwards, luminescent substrate is added, and the film is pressed into sheets for exposure in a darkroom.

The invention shows that the compound I inhibits the proliferation of lung cancer A549 cells by concentration dependence, and the result of the invention reveals that the compound I has the activity of resisting lung cancer proliferation and a possible action mechanism thereof, thereby providing a potential target molecule for the research and development of lung cancer chemotherapeutic drugs.

It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (7)

1. The application of a compound I in preparing a medicament for treating cancer is characterized in that the structural formula of the compound I is as follows:

the cancer is selected from lung cancer.

2. The use of a pharmaceutical formulation comprising a compound i for the manufacture of a medicament for the treatment of cancer, wherein the compound i has the structural formula:

the cancer is selected from lung cancer.

3. The use according to claim 2, wherein the formulation is selected from a liquid dosage form, a solid dosage form, a semi-solid dosage form or a gaseous dosage form.

4. The use of claim 3, wherein the liquid dosage form comprises an injection, infusion or oral liquid.

5. The use of claim 3, wherein the solid dosage form comprises a tablet, capsule, powder or granule.

6. The use of claim 3, wherein the semi-solid dosage form comprises an ointment or gel.

7. The use of claim 3, wherein the gaseous dosage form comprises an aerosol or a spray.

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