CN111789830B

CN111789830B - Application of 2- (2, 2-trifluoroethylene) -1, 3-dione compound in preparation of anti-lung cancer drugs

Info

Publication number: CN111789830B
Application number: CN202010803906.2A
Authority: CN
Inventors: 阳洁; 翁志强; 潘颖星; 莫孝成; 林棋; 吴伟; 黎骊; 莫小香
Original assignee: Minjiang University; Guangxi Medical University
Current assignee: Minjiang University; Guangxi Medical University
Priority date: 2020-08-11
Filing date: 2020-08-11
Publication date: 2023-04-28
Anticipated expiration: 2040-08-11
Also published as: CN111789830A

Abstract

The invention discloses application of a 2- (2,2, 2-trifluoroethylene) -1, 3-dione compound in preparation of an anti-lung cancer drug, and relates to application inventions in the field of medicines. The compound is preferably 1, 3-bis (4- (tert-butyl) phenyl) -2- (2,2, 2-trifluoroethylene) propane-1, 3-dione, which can inhibit the growth and proliferation of a human lung cancer cell line, bind to a specific region of TrxR1 protein of a human lung cancer cell and inhibit the functional activity of the TrxR1 protein, up-regulate the level of cellular ROS, finally lead to the blocking of the human lung cancer cell cycle to the G1 phase or the G2/M phase, promote the apoptosis or non-apoptotic death of the human lung cancer cell, and is a potential novel anti-lung cancer drug.

Description

Application of 2- (2, 2-trifluoroethylene) -1, 3-dione compound in preparation of anti-lung cancer drugs

Technical Field

The invention relates to the technical field of medicines, in particular to application of a 2- (2, 2-trifluoroethylene) -1, 3-dione compound in preparation of an anti-lung cancer medicine.

Background

Lung cancer is the first malignant tumor with incidence and death rate all living in the world and in China, and is also the main cause of cancer death, and seriously endangers the life and health of human beings. Lung cancer can be divided into two categories: non-small cell lung cancer (Non-small cell lung cancer, NSCLC) accounts for about 85% of lung cancer, and the Non-small cell lung cancer can be classified into adenocarcinoma, squamous carcinoma and large cell carcinoma; the other is small cell lung cancer (Small cell lung cancer, SCLC), which accounts for about 15% of lung cancer. At present, the treatment methods of lung cancer are mainly divided into: surgical treatment, chemotherapy, radiation therapy, molecular targeted therapy, and immunotherapy. Surgical treatment is an ideal treatment scheme for early non-small cell lung cancer, but most patients are in the middle and late stages of lung cancer when they visit, so that the surgical treatment is very limited. Thus, most patients can only select chemotherapy, radiation therapy, molecular targeted therapies, and immunotherapy. However, although chemotherapy, molecular targeted therapy and immunotherapy have remarkable therapeutic effects in the early stage of treatment, the problem of drug resistance of patients or no therapeutic response of partial patients inevitably occurs, and the treatment becomes a bottleneck limiting clinical efficacy. Therefore, there is an urgent need to find new anti-lung cancer targets and screen new anti-lung cancer drugs.

Many studies have shown that oxidative stress is an important mechanism for the pathogenesis of many diseases such as tumors, inflammation, etc. Oxidative Stress (OS) refers to an imbalance between in vivo oxidation and antioxidation, which tends to oxidize, and in vivo highly reactive molecules such as reactive oxygen species (Reactive oxygen species, ROS) are overproduced, which exceeds the rate of oxide removal, resulting in tissue damage. Most studies indicate that ROS are intimately involved in tumorigenesis and progression. On the one hand, the ROS rise can induce DNA mutation, exacerbate gene instability and abnormally activate a tumor promotion signal channel, so that the occurrence and malignant progress of tumors are promoted. On the other hand, however, excessive ROS production can damage intracellular molecules and organelles, ultimately leading to tumor cell death. Research reports that the grifola frondosa beta polysaccharide can induce the increase of ROS (reactive oxygen species) by activating oxidative stress so as to induce lung cancer cell apoptosis, capsaicin can induce the apoptosis of HepG2 liver cancer cells by oxidative stress, and oleanane triterpene CDDO-Me can inhibit the growth of prostate cancer cells and induce apoptosis by a ROS-dependent mechanism. These studies indicate that induction of oxidative stress and elevated ROS levels are one of the important strategies for anti-tumor therapy.

The research shows that the thioredoxin system plays an important role in the processes of oxidative stress, cell proliferation, apoptosis and the like. The Thioredoxin system consists of Thioredoxin reductase (Thioredoxin reductase, trxR), thioredoxin (Trx) and reduced nicotinamide adenine dinucleotide phosphate (Nicotinamide adenine dinucleotide phosphate, NADPH) together, regulating the redox state of the cell. Wherein TrxR is an NADPH dependent dimer selenase comprising a FAD domain, belonging to the family of pyridine nucleotide-disulfide oxidoreductase enzymes, having activity similar to glutathione reductase, which is one of the key enzymes of the glutathione redox cycle. Three isozymes are currently known for TrxR, namely TrxR1 (cytoplasmic type), trxR2 (mitochondrial type) and TrxR3 (expressed predominantly in testes, also known as TGR). Recent researches show that the TrxR1 is over-expressed in various human tumors such as lung cancer, breast cancer, gastric cancer and the like, and is closely related to the occurrence and development of the tumors and poor prognosis. Studies have shown that inhibition of TrxR1 causes an increase in ROS, leading to apoptosis. For example, curcumin analog CA6 causes increased ROS levels by inhibiting TrxR1, thereby inhibiting Akt and activating FoxO3a, ultimately inhibiting gastric cancer cell growth and inducing apoptosis. Therefore, trxR1 becomes an important potential molecular target in the development of new antitumor drugs.

1, 3-diketones are important backbones for many natural products, drugs, and other biologically relevant compounds, with a wide range of biological activities including antineoplastic, antioxidant, antibacterial, antiviral, and antifungal. Trifluoromethyl (CF) due to the unique nature of fluorine atoms ₃ ) The incorporation of groups into organic molecules tends to exhibit increased metabolic stability and improved pharmacokinetic properties. 2- (2, 2-trifluoroethylene) -1, 3-dione compounds are synthesized by introducing trifluoroethylene based on a 1, 3-dione structure, and part of the compounds have good bactericidal activity on cucumber downy mildew, wheat powdery mildew, corn rust and cucumber anthracnose. However, it is currently unclear whether 2- (2, 2-trifluoroethylene) -1, 3-dione compounds have anti-lung cancer activity.

Disclosure of Invention

The invention aims to provide an application of a 2- (2, 2-trifluoroethylene) -1, 3-dione compound, which has better inhibitory activity on human lung cancer cells and can be used as a potential anti-lung cancer drug.

The structure of the 2- (2, 2-trifluoroethylene) -1, 3-dione compound is as follows:

the applicant has found that the above compound 3e (chemical name: 1, 3-bis (4- (tert-butyl) phenyl) -2- (2, 2-trifluoroethylene) propane-1, 3-dione) and compound 3f (chemical name: 1, 3-bis (4-methoxyphenyl) -2- (2, 2-trifluoroethylene) propane-1, 3-dione), especially compound 3e, have significant anti-lung cancer effects by screening 20 2- (2, 2-trifluoroethylene) -1, 3-dione compounds, and thus the main object of the present application is to provide the use of the 2- (2, 2-trifluoroethylene) -1, 3-dione compounds of the above structure in the preparation of anti-lung cancer drugs.

Preferably, the medicament is a medicament for treating non-small cell lung cancer and/or small cell lung cancer.

Preferably, the medicament is a medicament for inhibiting growth and proliferation of lung cancer cells.

Preferably, the medicament is a medicament that promotes apoptosis or non-apoptotic death of lung cancer cells.

Preferably, the drug is a drug that blocks lung cancer cells to the G1 phase or the G2/M phase.

Preferably, the drug is a drug targeting TrxR1 protein. Specifically, the 2- (2, 2-trifluoroethylene) -1, 3-dione compound is TrxR1 protein that binds to human lung cancer cells and inhibits its functional activity.

Preferably, the lung cancer cells of the lung cancer are A549, H1299, HCC827, PC9, H460 or H1688.

The medicine is prepared from 2- (2, 2-trifluoroethylene) -1, 3-dione compound as main active component or one of the active components, and can contain pharmaceutically acceptable auxiliary materials according to requirements. The above-mentioned medicines may be any pharmaceutically acceptable dosage forms according to the different auxiliary materials and modes of administration, and these dosage forms may include: tablets, capsules, oral liquid, granules, medicinal granules, powder, suspension, powder, solution, injection and the like.

The beneficial effects of the invention are as follows:

the experimental verification shows that the 2- (2, 2-trifluoroethylene) -1, 3-dione compound, especially the compound 3e, can inhibit the growth and proliferation of a human lung cancer cell line, and through binding to TrxR1 protein of human lung cancer cells and inhibiting the functional activity thereof, the level of ROS in the cells is up-regulated, so that the cycle of the human lung cancer cells is finally blocked to the G1 phase or the G2/M phase, and the apoptosis or non-apoptotic death of the human lung cancer cells is promoted. Therefore, the compound can be used as a potential anti-lung cancer drug.

Drawings

FIG. 1, effect of 2- (2, 2-trifluoroethylene) -1, 3-dione compound (100. Mu.M) on cell viability of A549, H1299, HCC827, PC 9.

FIG. 2, effect of 2- (2, 2-trifluoroethylene) -1, 3-

dione compounds

3e and 3f on HCC827 and PC9 cell viability.

FIG. 3, effect of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e and cisplatin on A549, H1299, H460 and H1688 cell viability.

FIG. 4, effect of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e on the activity of TrxR1 in H1299 cell lysate.

FIG. 5, effect of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e on ROS levels in A549, H1299 cells.

FIG. 6, hoechst staining to observe the effect of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e and cisplatin on A549, H1299 nuclei.

FIG. 7, effect of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e on apoptosis rate of A549 and H1299 cells.

FIG. 8, effect of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e and cisplatin on A549, H1299 cell morphology (optical mirror X200).

FIG. 9, effect of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e and cisplatin on A549, H1299 cell morphology (HE staining X200).

FIG. 10, effect of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e on the sub-apparent microstructure of H1299, HCC827, H1688 cells (electron microscope X10000,. Times.20000).

FIG. 11, effect of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e on A549 and H1299 cell cycles.

Detailed Description

Example 12 screening of- (2, 2-trifluoroethylene) -1, 3-dione Compounds

The colorimetric experiments were performed using MTT.

The measurement principle is as follows:

succinate dehydrogenase in the mitochondria of living cells reduces exogenous MTT to water insoluble blue-violet crystalline Formazan (Formazan) and deposits in cells, whereas dead cells do not. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the OD value can be detected and read by an ELISA 490nm, so that the number of living cells can be indirectly reflected. The amount of MTT crystals formed is proportional to the number of living cells over a range of cell numbers.

The measuring method comprises the following steps:

1) And (3) in the primary screening stage, four lung cancer cells of A549, H1299, HCC827 and PC9 are respectively inoculated into 96-well plates, the number of the cells is 3000-8000 per well, the number of cell suspension is 100 mu L per well, 3 compound wells are arranged, and the cells are cultured in an incubator for 24 hours to adhere to the walls. After the cells were attached, 100. Mu.L of each compound at a concentration of 200. Mu.M was added to each of the drug treatments, and the final concentration of the cells was 100. Mu.M, and the control group was added to the medium, followed by further culturing for 24 hours. After adding 20. Mu.L MTT (5 mg/ml) per well and incubating for 4 hours in an incubator, the supernatant was discarded, 150. Mu.L DMSO per well was added, and the mixture was placed on a shaker and shaken horizontally for 5 minutes, and OD was detected and read at 490nm using an ELISA. The preliminary screening stage is a preliminary comparative screening of 20 2- (2, 2-trifluoroethylene) -1, 3-dione compounds.

2) In the re-screening stage, HCC827 and PC9 cells are inoculated into 96-well plates respectively, after the cells are attached, a series of primary screening compounds are added into a drug treatment group, and after preliminary experiments, the HCC827 and PC9 cells are added into the primary screening compounds with final concentrations of 80 mu M, 40 mu M, 20 mu M, 10 mu M and 5 mu M. The rescreening stage compares the anticancer activity of the various concentrations of the primary screening compound.

As shown in FIG. 1 and Table 1, the primary screening results showed that 20 2- (2, 2-trifluoroethylene) -1, 3-dione compounds acted on A549, H1299, HCC827, PC9 cells at a concentration of 100. Mu.M for 24 hours, respectively, and exhibited different cell growth inhibitory effects. 4. The inhibition rates of the compounds 3e, 3a, 3p and 3f on A549 cells are 89.5+/-2.8%, 88.8+/-1.0%, 87.7+/-2.1% and 87.4+/-1.2% respectively, and the inhibition rates of the other 16 compounds on A549 cells are 10% -80%; the inhibition rate of 6 compounds 3e, 3m, 3f, 3a, 3k and 3p on H1299 cells is 93.9+/-1.6%, 92.1+/-2.2%, 90.8+/-5.2%, 86.9+/-7.3%, 86.0+/-6.2% and 85.9+/-7.6%, and the inhibition rate of the other 14 compounds on H1299 cells is 10% -80% or the proliferation promoting effect is achieved; the inhibition rates of 3 compounds 3e, 3g and 3f on HCC827 cells are respectively 89.0+/-4.2%, 87.8+/-4.1% and 85.4+/-7.0%, and the inhibition rates of the rest 17 compounds on HCC827 cells are 0% -80% or have proliferation promoting effect; the inhibition rates of 3 compounds 3e, 3f and 3m on PC9 cells are respectively 90.9+/-4.0%, 90.0+/-4.4%, 84.3+/-16.0%, and the inhibition rates of the rest 17 compounds on PC9 cells are 0% -60% or have proliferation promoting effect. The results show that the inhibition rate of the two

compounds

3e and 3f on four lung cancer cells A549, H1299, HCC827 and PC9 is more than 80%, and the inhibition rate of the compound 3e on four cells is the highest. Therefore, it is required to further expand the kinds of lung cancer cells, and further

re-screen compounds

3e and 3f among 2- (2, 2-trifluoroethylene) -1, 3-dione compounds and confirm the anti-lung cancer activity.

As shown in FIG. 2, the re-screening results showed that the survival rate of HCC827 and PC9 cells decreased significantly with the increase of the concentration of 2- (2, 2-trifluoroethylene) -1, 3- dione compound 3e 0. Mu.M-80. Mu.M, and the survival rate of HCC827 and PC9 cells was only about 20% or less at the concentration of 80. Mu.M, and the IC of 3e on HCC827 and PC9 cells was observed ₅₀ 26.29.+ -. 0.37. Mu.M and 19.52.+ -. 0.58. Mu.M, respectively. Whereas at a concentration of 3f 80. Mu.M of the 2- (2, 2-trifluoroethylene) -1, 3-dione compound, the survival rates of both HCC827 and PC9 cells were above 50%, i.e. the IC of 3f on HCC827, PC9 cells ₅₀ Greater than 80 μm. The above results indicate that the anti-lung cancer activity of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e is superior to that of the same typeCompound 3f (P)<0.05)。

Table 12 Structure and cytostatic Rate of- (2, 2-trifluoroethylene) -1, 3-dione Compounds

(n＝3，

)/>

/>

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Example 2 2 inhibition of lung cancer cells cultured in vitro by 2, 2-trifluoroethylene-1, 3-dione compound 3e was performed using an MTT colorimetric assay.

The measuring method comprises the following steps:

1) Six lung cancer cell lines A549, H1299, HCC827, PC9, H460, H1688 were each cultured in RPMI1640 medium containing 10% FBS and 1% penicillin/streptomycin mixture at 37℃and 5% CO ₂ Culturing in a constant temperature cell incubator. The experimental operations of cell liquid exchange, digestion passage, seed plate, drug addition and the like are carried out in a sterile super clean bench, and the liquid exchange is carried out at proper time according to the proliferation speed and the density of the cells.

2) The anti-lung cancer activity of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e is detected by adopting an MTT method, six lung cancer cells of A549, H1299, HCC827, PC9, H460 and H1688 are respectively inoculated into 96-well plates, the number of the cells is 3000-8000 per well, the cell suspension is 100 mu L per well, 3 compound wells are arranged, and the cells are cultured in an incubator for 24 hours to adhere the cells.

3) Compound 3e was added to HCC827 and PC9 cells at final concentrations of 80. Mu.M, 40. Mu.M, 20. Mu.M, 10. Mu.M, 5. Mu.M, A549, H1299, H460 cells at final concentrations of 25. Mu.M, 20. Mu.M, 15. Mu.M, 10. Mu.M, 5. Mu.M, H1688 cells at final concentrations of 80. Mu.M, 40. Mu.M, 20. Mu.M, 10. Mu.M, 5. Mu.M, and cisplatin was selected as a positive control for anti-tumor effect, and the control was added to the medium for further culture for 24 hours.

4) mu.L MTT (5 mg/mL) was added to each well, after incubation in an incubator for 4 hours, the supernatant was discarded, 150. Mu.L MSO was added to each well, and the mixture was placed on a shaker and shaken horizontally for 5 minutes, and OD values were detected and read at 490nm using an ELISA. Recording and storing data, calculating IC by probit regression method in SPSS software ₅₀ . Data are expressed as mean ± standard deviation (x±sd) of triplicate experiments, comparisons between two groups using t-test, P<A difference of 0.05 is statistically significant.

MTT test results show that the 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e shows obvious cell growth inhibition effect on six lung cancer cells after 24 hours of administration, and concentration dependence exists on the growth inhibition effect of the cells. In particular, compound 3e IC in comparison with A549, H1299, H460, H1688 cells ₅₀ 14.94+ -0.76 μM, 8.49+ -0.55 μM, 21.84+ -1.68 μM, 30.04+ -6.92 μM, respectively, whereas the positive control agent cisplatin was IC on A549, H1299, H460, H1688 cells ₅₀ 32.25.+ -. 5.93. Mu.M, 41.47.+ -. 6.55. Mu.M, 64.40.+ -. 9.69. Mu.M, 85.28.+ -. 30.34. Mu.M, respectively. By the comparison, compound 3e was tested for IC on the four lung cancer cells ₅₀ IC smaller than cis-platinum ₅₀ And the differences are statistically significant (P<0.05 The anti-lung cancer activity of the compound 3e is better than that of cisplatin. The results are shown in FIG. 3 and Table 2.

TABLE 2 IC of active Compound 3e and cisplatin on lung cancer cells ₅₀

(n＝3，`x±s)

In comparison with the cisplatin group, ^* P<0.05; "-" represents undetected.

Example 32 molecular docking of- (2, 2-trifluoroethylene) -1, 3-dione Compound 3e with TrxR1 protein and Activity verification

MOE molecular docking software and DTNB experiments were used.

The measurement principle is as follows:

1) Molecular docking (Molecular Docking) is a process of mutual recognition between ligands and receptors by energy and geometric matching, and mainly includes electrostatic action, hydrogen bonding action, hydrophobic action, van der waals force, etc. In recent years, with the rapid development of computer-aided drug design technology, molecular docking technology has become one of the important methods for structure-based drug design and computer-aided drug design.

2) DTNB is an Ellman reagent which is used in colorimetry to determine thiol groups in biological samples. It is easy to dissolve in water, in the presence of mercapto compound, colorless DTNB will be converted into yellow 5-mercapto-2-nitrobenzoic acid, and then OD value is read at 412nm in enzyme label instrument to calculate inhibition rate, which can reflect TrxR1 protein activity indirectly.

The measuring method comprises the following steps:

1) The 2D structures of 20 fluorine-containing 2- (2, 2-trifluoroethylene) -1, 3-dione compounds were each drawn using the chemical structural software chembiosdraw and converted to the lowest energy 3D structure, and then all the compound structures were imported into the molecular docking software MOE to form the MDB file. The protein structure with code 3qfb was downloaded from the PDB website and pretreated using Discovery Studio software to remove water molecules, heterochains, etc., but retain its ligand structure. The pretreated protein structure was opened using MOE software, hydrogen atoms and charges were added to the protein using protonate3D, and energy minimization was performed on the protein using energyinimize. According to the ligand expansion principle, the ligand is selected, and an active site is established with the ligand as the center and the radius of 8 angstroms.

2) The original ligand was molecularly docked to the protein, the lowest RMSD and the conformation closest to the experimental structure were selected, and the optimal combination of scoring and optimization functions was determined. The compound and the protein are prepared for molecular docking by dock, namely a receptor is selected, a docking site is an active site established before selection, a ligand is a compound MDB file established before selection, and a scoring function and a function determined before optimization function selection are performed for docking.

3) Cells were plated in 100mm dishes overnight at about 90% cell density, 1mL of cell lysate was added and the cells were collected. The collected cells were centrifuged (12000 rpm/min,10 min) at 4℃and the supernatant was collected for enzyme activity detection. EDTA powder was dissolved in 100mM phosphate buffer (final EDTA concentration of 2 mM) to prepare PE buffer. NADPH powder was dissolved in PE buffer as described above (formulated at 1.66 mg/mL), and DTNB powder was dissolved in PE buffer (formulated at 3.97 mg/mL). To the 96-well plate, 40. Mu.L of NADPH solution was added, followed by 2. Mu.L of the supernatant after centrifugation, and 0.5. Mu.L of the drug at different concentrations was added, and incubated at room temperature for 2 hours. The 96-well plate was rapidly added with 40. Mu.L of DTNB solution, OD was read at 412nm on a microplate reader, and inhibition was calculated.

In the molecular docking result, E-conf is conformational energy, E-place is placement energy, E-score1 is first scoring, E-refine is optimizing energy, E-score2 is second scoring, S value is last scoring, and smaller S value represents stronger affinity and more stable conformation. The results are shown in Table 3, where the S value of active compound 3e is the lowest (-32.4585) and lower than that of the positive control group curcumin (-25.7874), indicating that 3e may have stronger binding with TrxR1 and more stable conformation. The TrxR1 activity results (as shown in fig. 4) show that compound 3e can inhibit TrxR1 activity in a concentration-dependent manner.

TABLE 3 results of the docking of trifluoroethylene-containing 2- (2, 2-trifluoroethylene) -1, 3-dione compounds with TrxR1 molecules

Example 42 Effect of- (2, 2-trifluoroethylene) -1, 3-dione Compound 3e on ROS levels in lung cancer cells

1. Performed using a flow cytometer.

The measurement principle is as follows:

DCFH-DA is a nonpolar substance that can cross the cell membrane into the cell. After entering cells, DCFH with polarity is formed, which can be matched with H in cells ₂ O ₂ The reaction produced DCF. The latter can be used to measure fluorescence intensity in a flow cytometer for detecting ROS production.

The measuring method comprises the following steps:

cell count 1X 10 ⁶ The cell density of each cell was inoculated at 25cm ² In a cell culture flask. After 24h adherence, adding medicine for 3h, then collecting cells, transferring cell suspension to a 15ml centrifuge tube, centrifuging for 1000r 5min, discarding supernatant, adding one-tube PBS to resuspend for 1000r 5min, discarding supernatant, adding 1ml PBS to blow and mix uniformly, transferring to a 1.5ml centrifuge tube, centrifuging for 1000r 5min, discarding. The diluted DCFH-DA staining solution (the final concentration of the DCFH-DA is 10 mu M by diluting the DCFH-DA with serum-free culture solution according to the ratio of 1:1000 in advance) is added into the cells, the cells are uniformly mixed by blowing, and the cells are incubated for 20min in a cell culture box at 37 ℃. The centrifuge tube was shaken up and down 3 times during staining to allow staining to be complete, and the cells were washed three times to remove DCFH-DA that did not enter the cells sufficiently. The test tube is marked, the cells are filtered, and a flow cytometer is used for detecting the influence of the 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e on the ROS level in lung cancer A549 and H1299 cells, DMSO is used as a solvent control, and the active oxygen positive induction drug Rosup is used as a positive control. The level of intracellular active oxygen was analyzed according to the magnitude of its fluorescence signal intensity.

As shown in FIG. 5A, compared with the DMSO negative control group, the concentration of the 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e on the A549 and H1299 cells was increased (7.5. Mu.M, 15. Mu.M, 30. Mu.M) (4. Mu.M, 8. Mu.M, 16. Mu.M) 3H, the ROS levels in the A549 and H1299 cells were also gradually increased (1.72.+ -. 0.11, 1.78.+ -. 0.10, 1.96.+ -. 0.24) (1.08.+ -. 0.02, 1.11.+ -. 0.03, 1.16.+ -. 0.02) (P < 0.05), and the level of ROS in the A549 cells induced by 30. Mu.M of the 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e was increased, the level of ROS in the H1299 cells induced by 16. Mu.M was increased, which was comparable to that in the positive control group (P < 0.05). The addition of the antioxidant N-Acetyl-L-cysteine (i.e., ROS scavenger, N-Acetyl-L-cysteine, NAC) reduced the increase in ROS in A549 and H1299 cells (1.39.+ -. 0.26, 1.07.+ -. 0.07) (P < 0.05) caused by 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e compared to compound 3e treatment group. Furthermore, antioxidant NAC reduced ROS levels (0.80±0.17, 0.96±0.09) in a549 and H1299 cells compared to DMSO negative control, but the differences were not statistically significant (P > 0.05); compound 3e increased ROS levels (1.39±0.26, 1.07±0.07) in a549 and H1299 cells after treatment with anti-oxidant NAC (P < 0.05) compared to the anti-oxidant NAC treatment group (0.80±0.17, 0.96±0.09).

2. Performed using a fluorescence microscope.

The measurement principle is as follows:

DCFH-DA itself has no fluorescence, can freely pass through cell membranes, and can be hydrolyzed by intracellular esterases to generate DCFH after entering cells. Whereas DCFH cannot penetrate the cell membrane, thus making the probe easily loaded into the cell. Intracellular reactive oxygen species can oxidize non-fluorescent DCFH to produce fluorescent DCF. The level of intracellular active oxygen can be known by detecting the fluorescence of DCF. The stronger the green fluorescence indicates higher ROS levels, and the weaker the green fluorescence indicates lower ROS levels.

The measuring method comprises the following steps:

cells were 3X 10 per well ⁴ Individual cell densities were seeded in 24-well plates. After 24h adherence, the cell culture solution is discarded after 3h of drug addition, 500 μl of diluted DCFH-DA staining solution (the final concentration is 10 μM by diluting DCFH-DA with serum-free culture solution at 1:1000 in advance) is added into each well, so as to fully cover the cells, and the cells are incubated in a cell incubator at 37 ℃ for 20min. The cells were washed three times with serum-free cell culture medium to sufficiently remove DCFH-DA that did not enter the cells. Observed under a fluorescence microscope and photographed.

Fluorescence microscopy observations showed (FIG. 5B) that after 3H of action of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e, the green (white in the figures) fluorescence intensity of A549, H1299 cells gradually increased, i.e., 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e enhanced ROS levels in lung cancer cells A549, H1299; while the green fluorescence intensity enhancement by 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e can be significantly reduced upon addition of the antioxidant NAC, i.e., the antioxidant NAC antagonizes the elevated ROS levels by 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3 e.

The above results indicate that 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e can cause elevated ROS levels in lung cancer cells a549, H1299 cells, while antioxidant NAC can antagonize elevated ROS levels in a549, H1299 cells caused by 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3 e.

Example 52 influence of- (2, 2-trifluoroethylene) -1, 3-dione Compound 3e on apoptosis of Lung cancer cells

1. The staining was performed using Hoechst.

The measurement principle is as follows:

hoechst33342 is a non-intercalating fluorescent dye. They bind to DNA in the minor groove of the DNA polyat sequence-rich region in living cells. Either living or fixed cells can ingest the dye from a low concentration solution, thereby staining the nuclei. When excited by ultraviolet light of a fluorescence microscope, hoechst-DNA emits bright blue fluorescence.

The measuring method comprises the following steps:

1) Cells were grown overnight in six well plates to give a cell fusion of approximately 50% -80%. After 24 hours from the administration of the cells, the culture broth was drained, and 0.5mL of a fixative was added and the mixture was fixed for 10 minutes or longer. The fixative was removed and washed twice with PBS for 3 minutes each to drain the solution. The washing machine is rocked for several times by a cradle or manually. mu.L of Hoechst33342 staining solution was added and stained for 10 minutes, and the mixture was shaken by shaking or manual shaking for several times. Wash twice with PBS for 3 minutes each.

2) The photographs were observed under a fluorescence microscope.

As a result, as shown in FIG. 6, in contrast to the control group, the nuclei were concentrated after the compound 3e and cisplatin acted on the A549 cells, and a blue highlighting phenomenon (the light white color is shown in the black-white chart), and a part of the cells appeared to have nuclei disintegrated, which was consistent with the phenomenon of apoptosis, suggesting that the 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e induced apoptosis of the A549 and H1299 cells.

2. The method is carried out by using an Annexin V/PI double-dyeing method.

The measurement principle is as follows:

annexin V is a Ca ²⁺ The dependent phospholipid binding proteins have the property of easily binding to phospholipids such as Phosphatidylserine (PS), and have a high affinity for PS. Thus, the protein can act as a sensitive probe to detect PS exposed on the surface of the cell membrane. However, PS transfer outside the cell membrane is not unique to apoptosis and can also occur in cell necrosis. Furthermore, apoptotic cells are resistant to all dyes used for cell activity identification, such as PI, and necrotic cells are not. The DNA of cells with damaged cell membranes can be stained by PI to generate red fluorescence, while cells with intact cell membranes do not generate red fluorescence. Thus, PI does not stain at the early stages of apoptosis without a red fluorescent signal. Normal living cells are similar to this. On the scatter plot of the bivariate flow cytometer, the lower left quadrant shows living cells as (FITC-/PI-); the upper right quadrant is non-viable cells, i.e. cells late in apoptosis and necrotic cells, being (fitc+/pi+); while the lower right quadrant is early apoptotic cells, revealing (fitc+/PI-); while the upper left quadrant is the detection error within the allowed range.

The measuring method comprises the following steps:

1) Two strains of A549 and H1299 cells were cultured according to a ratio of 1X 10 ⁶ Cell densities of the cells were inoculated to 25cm ² In a cell culture flask. After 24h adherence, DMSO was used as solvent control and cisplatin as positive control. Adding the medicines for 24h and 48h.

2) Collecting supernatant into centrifuge tube, digesting cells in bottle with pancreatin without EDTA, adding serum-containing culture medium to stop digestion, collecting cells into the front centrifuge tube, cleaning culture bottle with culture medium again and collecting residual cells into the same centrifuge tube, centrifuging at 1000rpm/min for 10min, adding 1mL of PBS into centrifuge tube, blowing cells uniformly and transferring to 1.5mL of EP tube, centrifuging at 600g for 5min. The centrifugation was resuspended in 1mL of PBS. Then, 100. Mu.L of 1 Xbinding buffer (diluted 1X with pure water) was added, and 5. Mu.L of Annexin V-PE and 5. Mu.L of 7-AAD were added thereto, and the mixture was protected from light at room temperature for 15 minutes. 200 mu L of 1X Binding buffer is added, a cell filter screen is used, and the detection is carried out on the machine.

As shown in FIG. 7, when 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e was applied to A549, H1299 cells at a low concentration (7.5. Mu.M or 4. Mu.M) for 24 hours or 48 hours, the apoptosis rate of A549, H1299 cells was not significantly affected or was increased to some extent (DMSO vs 3e,24 hours: 5.86.+ -. 0.01% vs 6.66.+ -. 0.18%, 5.43.+ -. 0.35% vs 6.58.+ -. 0.77%, P >0.05;48H: 3.85.+ -. 0.62% vs 7.13.+ -. 0.40%, 2.13.+ -. 0.28% vs 4.61.+ -. 0.31%, P <0.05 and P >0.05 respectively), whereas medium (15. Mu.M or 8. Mu.M), high (30. Mu.M or 16. Mu.M) concentrations of 2- (2, 2-trifluoroethylene) -1, 3-dione compounds 3e, after 24H, 48H, respectively, of A549, H1299 cells, respectively, can cause an increase in the apoptosis rate of the A549 cells (DMSO vs 3e,24H: medium concentration 5.86+ -0.01% vs 8.69+ -0.64%, high concentration 5.86+ -0.01% vs 12.87+ -2.60%, P <0.05, 48H medium concentration 3.85+ -0.62% vs 70.06+ -2.55%, high concentration 3.85+ -0.62% vs 94.69+ -1.65%, P < 0.05) and H1299 increase apoptosis rate (DMSO vs 3e,24H medium concentration 5.43+ -0.35% vs 6.14+ -1.31%, high concentration 5.43+ -0.35% vs 9.86+ -0.28%, P >0.05 and P <0.05, respectively, 48H medium concentration 2.13+ -0.28% vs 24.75+ -2.34%, high concentration 2.13+ -0.28% vs 45.90+ -5.29%, P < 0.05). These results indicate that 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e can promote apoptosis of a549, H1299 cells in a concentration-dependent manner.

In addition, compared with the DMSO group, after 30 mu M and 40 mu M positive control cisplatin are treated for 24 hours respectively, the apoptosis rate is increased (DMSO vs cisplatin: 5.86+ -0.01%vs 21.34+ -0.34%, 5.43+ -0.35%vs 20.92+ -3.88%, P < 0.05) and after the cisplatin is treated for 48 hours respectively, the apoptosis rate is further increased significantly (DMSO vs cisplatin: 3.85+ -0.62 vs 42.45+ -4.12%, 2.13+ -0.28 vs 70.55+ -2.81%, P < 0.05). Notably, compound 3e induced apoptosis more significantly than cisplatin (70.06 ±2.55% vs 42.45±4.12%, 94.69±1.65% vs 42.45±4.12%, and P < 0.05) after treatment of a549 cells with a medium and high concentration (15 μm, 30 μm) of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e for 48h compared to the 30 μm positive control cisplatin-treated group. These results show that 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e has a remarkable effect of inducing apoptosis of lung cancer cells.

In addition, compared with the DMSO group, after 1mM of antioxidant NAC is used for treating the A549 cells and the H1299 cells for 24 hours and 48 hours, the apoptosis rate of the H1299 cells is not obviously influenced (P is more than 0.05); in addition, although the apoptosis rate of A549 was increased (P < 0.05), the apoptosis rate was very low (DMSO vs NAC,24h: 5.86.+ -. 0.01% vs 8.08.+ -. 0.67%;48h: 3.85.+ -. 0.44% vs 7.73.+ -. 0.11%). However, the 1mM antioxidant NAC in combination with high concentration compound 3e significantly reduced the apoptosis rate of lung cancer cells (P < 0.05) after 48h treatment of both A549 and 1299 cells compared to the high concentration compound 3e alone treatment group. This suggests that the antioxidant NAC may partially antagonize the pro-apoptotic effects of compound 3 e. Meanwhile, compared with an antioxidant NAC single treatment group, after the NAC is combined with a high-concentration compound 3e to treat A549 and 1299 cells for 48 hours, the apoptosis rate of the two lung cancer cells can be obviously increased, and further the effect of promoting the apoptosis of the lung cancer cells by the 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e is suggested.

Example 62 Effect of- (2, 2-trifluoroethylene) -1, 3-dione Compound 3e on non-apoptotic death of Lung cancer cells

1. Using an optical microscope.

The measurement principle is as follows:

the change in cell morphology can be visually observed by an optical microscope to investigate the effect of compound 3e on non-apoptotic death of lung cancer cells.

The measuring method comprises the following steps:

1) Cells were seeded in 24-well plates and after 24h adherence, drug was added. Cell morphology was observed under an optical microscope at various time points and photographed according to the time and intensity of cavitation of cells.

2) Adherent cells were washed 2 times with PBS, digested with pancreatin, counted 5X 10 per well ⁴ The cells were seeded in 6-well plates with autoclaved coverslips, supplemented with medium to 1.5mL, and incubated in an incubator for 24h. The solution was washed with PBS for 3 times,different concentrations of drug were given and incubation was continued for 24h. The medium was discarded, fixed with 95% ethanol for 20min, and washed with PBS 2 times, each for 1min. Hematoxylin staining for 5min, tap water washing and pure water washing. Eosin staining for 5min, washing with tap water, and washing with pure water. Sealing piece: blow-drying, and sealing the neutral resin. And (5) observing and photographing under a light mirror.

As a result, as shown in FIG. 8, 15. Mu.M of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e treated A549 cells, the appearance of vacuoles (arrow) was seen for 6 hours, and the nuclei remained intact; the 12h cavitation is more serious, basically occupies the cytoplasmic space, and the cell nucleus is not changed yet; whereas 30. Mu.M cisplatin treatment of A549 cells for 12h did not cause cavitation. In addition, 8. Mu.M 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e treated H1299 cells, and the cells were seen to appear vacuolated (indicated by arrow) and cell debris in 9 hours, with vacuoles of different sizes and surrounding mainly the nucleus; the cytoplasm is dissolved in 12 hours, the cell structure is incomplete, the cells are seriously damaged, and more cell fragments appear; many cells float and die for 24 hours; whereas 40. Mu.M cisplatin treated H1299 cells for 24H had no cytoplasmic vacuolation. The above results indicate that 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e affects the morphology of a549 and H1299 cells, inducing the cytoplasm to produce a large number of vacuoles which surround mainly the nucleus, but the nucleus remains intact.

As shown in FIG. 9, the HE staining results show that the A549 cells of the DMSO control group are long fusiform or triangular, the H1299 cells are polygonal or circular, the A549 and H1299 cells are complete in morphology, the cytoplasm is rich, and no vacuoles exist. After the 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e is acted for 12 hours, empty bubbles appear in cytoplasm of A549 and H1299, the number of the empty bubbles is large (indicated by arrows), and the cell nucleus is unchanged. After 24H of action of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e, the cytoplasm of A549 cells decreased, and part of H1299 cells had shrunken, and the nuclei of both A549 and H1299 cells had not been changed. After 24 hours of cisplatin action, A549 and H1299 cells shrink, cytoplasms are reduced, cell nucleus is collected, and cytoplasms are not vacuolated. HE staining results showed that, in agreement with the above-described optical results, 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e induced cavitation of lung cancer cells.

2. Using a transmission electron microscope.

The measurement principle is as follows:

the transmission electron microscope was able to observe the cell ultrastructure to investigate the effect of compound 3e on non-apoptotic death of lung cancer cells.

The measuring method comprises the following steps:

1) Cells were plated at 3X 10 per well ⁵ The individual cells were seeded in six well plates, 3 groups of cells each, including control group, drug treatment group 1, drug treatment group 2 (drug treatment groups 1 and 2 have the same concentration of drug and different treatment times), 2 wells per group. After 24H cell attachment, the drug-treated groups H1299, HCC827, H1688 cells were added with compound 3e 8. Mu.M, 26. Mu.M, 30. Mu.M, respectively, at the respective IC50 concentrations, and the control group was added with medium.

2) According to the time and intensity of cavitation of each cell, H1299 cells were collected by digestion with pancreatin at 12H and 24H, HCC827 cells at 6H and 12H, H1688 cells at 24H and 48H, centrifuged, washed with PBS 1 time, centrifuged again, gently blown with 1mL PBS uniformly, transferred to 1.5mL EP tube, and centrifuged. Slowly adding precooled 3% glutaraldehyde along the pipe wall, fixing for more than 2h at 4 ℃ for 3 times, washing with PBS for 10-15min each time, fixing with 1% osmium acid for 1-2h each time, washing with PBS for 3 times, washing with PBS for 10-15min each time, carrying out gradual dehydration with ethanol-acetone, wherein 50% ethanol is 1 time (4 ℃), 70% ethanol is 1 time (4 ℃), 90% ethanol is 1 time, 90% acetone is 1 time, 100% acetone is 3 times, dehydrating for 10-15min each, soaking with acetone and embedding medium, wherein acetone is 1:1 for more than 2h, acetone is 1:2 for more than 1-3h or overnight, fully soaking with pure embedding medium for 2h or overnight, embedding with pure embedding medium epoxy 618, and then carrying out polymerization (40-48-60 ℃ for 15h-2h-24 h), repairing, slicing, dyeing, carrying out observation under a transmission electron microscope, and photographing.

As shown in fig. 10, under the action of the compound 3e, mitochondrial swelling appears in H1299 cells at early stage (see fig. 10B), vacuoles appear in the cells, but the morphology of cell nuclei is not changed, vacuoles at late stage (see fig. 10C) are more, the size of the vacuoles around the cell nuclei is different (shown by arrows), the cell nuclei are still complete, the nuclear membrane is complete, the cell membranes are still complete, and the morphology of the cells of the control group (see fig. 10A) is not changed; HCC827 cells also show vacuolation (arrow) at early stage (see fig. 10E), vacuoles are irregular, nuclear morphology is not changed, more vacuoles (arrow) are still irregular at late stage (see fig. 10F), some vacuoles are very large and some vacuoles are very small, and control group (see fig. 10D) cell morphology is not described above; h1688 cells did not see vacuoles early (see FIG. 10H), but showed severe mitochondrial swelling, balloon-like changes (arrow), vacuolation (arrow) in late (see FIG. 10I), and control group (see FIG. 10G) cell morphology did not appear. Notably, cytoplasmic aggregated vacuoles appear as a monolayer, with substantially no subcellular organelle structures within the vacuoles, and fusion between portions of the vacuoles occurs, forming larger vacuoles, consistent with the morphological characteristics of giant bubble death.

Example 72 influence of- (2, 2-trifluoroethylene) -1, 3-dione Compound 3e on the lung cancer cell cycle

Performed using a flow cytometer.

The measurement principle is as follows:

when the cell cycle progress changes periodically, the DNA content in the cells changes, for example, the DNA content in the G0 and G1 phases is diploid, the DNA content in the G2 phase is tetraploid, and when external stimulus acts on the DNA, DNA fracture is caused, and the content of the DNA is also changed. Propidium Iodide (PI) is a dye that can be inserted between bases to bind to DNA, and fluoresces under excitation light of a certain wavelength, the fluorescence intensity of which is proportional to the DNA content.

The measuring method comprises the following steps:

1) The effect of compound 3e on lung cancer a549 and H1299 cell cycle was examined by flow cytometry using DMSO as solvent control. The cells were packed in 1X 10 cells ⁶ Cell densities of the cells were inoculated to 25cm ² In a cell culture flask. After the wall is attached for 24 hours, adding the medicine for 24 hours.

2) Digesting cells with pancreatin, adding a serum-containing culture medium after digestion, stopping digestion, blowing the cells, transferring the cell suspension to a 15mL centrifuge tube, centrifuging at 1000rpm/min for 5min, discarding the supernatant, adding a tube of PBS for resuspension, centrifuging at 1000rpm/min for 5min, discarding the supernatant, adding 1mL of PBS for blowing and mixing uniformly, transferring to a 1.5mL centrifuge tube, centrifuging at 1000rpm/min for 5min, discarding 900 mu L, and resuspension the sediment in residual liquid by a light bomb wall. The cells were mixed with a 100. Mu.L gun, 1mL of 75% ethanol at-20deg.C was slowly added to the cells, gently blown (to avoid cell clumping) while adding, and fixed overnight at-20deg.C.

3) On the day of detection, the cells were centrifuged at 400rpm/min for 10min, ethanol was removed, the walls of the flick tube were loosened, 1mL of PBS was added to the cells, the mixture was blown and mixed well, the centrifugation was performed at 400rpm/min for 10min, and 900. Mu.L of the supernatant was removed. The cells were mixed with a 100. Mu.L gun, 500. Mu. L DNA staining solution was added and vortexed for 5-10 seconds. Incubation for 30min at room temperature in dark place, taking out again in 15min midway, and mixing by vortex. And (5) cell filtering net, and detecting on the machine.

As a result, as shown in FIG. 11, after each treatment of A549 and H1299 cells with compound 3e for 24 hours, the ratio of cells in the G1 phase of A549 cells was increased and concentration-dependent (P < 0.05), the ratio of cells in the G2/M phase of H1299 cells was increased and concentration-dependent (P < 0.05), compared with the DMSO group, suggesting that compound 3e had a regulatory effect on the cell cycle of A549 and H1299, and blocking the cells in the G1 phase and G2/M phase, respectively.

NAC reduced the G1 phase cell fraction of A549 cells (P < 0.05) but had no significant effect on the H1299 cell cycle (P > 0.05) after 24H treatment of A549 cells, H1299 cells with 1mM antioxidant NAC compared to the DMSO group. In addition, the ratio of cells in the G1 phase and the ratio of cells in the G2/M phase of A549 cells and H1299 cells were significantly reduced (P < 0.05) after treatment of A549 cells, H1299 cells for 24H with 1mM antioxidant NAC in combination with high concentration (30. Mu.M) of compound 3e, compared with the high concentration (30. Mu.M) of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e group. Furthermore, the proportion of cells in the G1 phase (P < 0.05) was significantly increased after 24h treatment of a549 cells with 1mM antioxidant NAC in combination with high concentration (30 μm) of compound 3e compared to the 1mM antioxidant NAC group. The above results indicate that 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e has a modulating effect on the A549 and H1299 cell cycle, whereas antioxidant NAC antagonizes the modulating effect of 2- (2, 2-trifluoroethylene) -1, 3-dione compound 3e on the cell cycle.

Claims

Use of a 2- (2, 2-trifluoroethylene) -1, 3-dione compound in the manufacture of a medicament for treating lung cancer, wherein the 2- (2, 2-trifluoroethylene) -1, 3-dione compound has the structure:

。
2. the use of claim 1, wherein the medicament is a medicament for the treatment of non-small cell lung cancer and/or small cell lung cancer.
3. The use of claim 1 wherein the medicament is a medicament comprising a 2- (2, 2-trifluoroethylene) -1, 3-dione compound and other pharmaceutically acceptable excipients.