TWI694990B - Pharmaceutical composition containing a novel compound for selectively killing oral cancer cells and use of the compound thereof - Google Patents

Abstract

A novel sulfonyl chromen-4-ones compound and a pharmaceutical composition having the compound for selectively killing oral cancer cells are provided. The pharmaceutical composition includes a sulfonyl chromen-4-ones as an active ingredient and a pharmaceutically acceptable carrier or salt. The compound is 2-naphthalen-1-ylmethyl-3-(toluene-4-sulfonyl)chromen-4-one and has an effect of selectively killing the oral cancer cells. A use of a sulfonyl chromen-4-ones compound for manufacturing a medicament to selectively killing oral cancer cells and a use of a sulfonyl chromen-4-ones compound for manufacturing a cancer treatment medicament mediated by oxidative stress are also provided.

Description

Pharmaceutical composition containing novel compound for selectively killing oral cancer cells and use of the compound

The present invention relates to a novel compound, and particularly to a pharmaceutical composition containing the compound as an active ingredient for selectively killing oral cancer and the medical use of the compound.

Cancer has become the top ten cause of death among Chinese people, and the treatment or control of cancer is increasingly important. Cancer cells are caused by malignant normal cells, so they still retain some metabolic properties of normal cells. In recent years, although many anti-cancer drugs have been reported, however, due to the similarity between cancer cells and normal cells, anti-cancer drugs cause damage to normal cells in addition to killing cancer cells. Finding drugs that can selectively kill cancer cells has become an important issue.

Functional chromones are one of the main components of naturally oxidized heterocycles. It has been reported that chromone has anti-inflammatory and anti-cancer functions. For example, the isoform of 2-styrylchromone has been shown to have anticancer effects on K562 lymphoma cells, HCT-15 colorectal cancer cells, A549 lung cancer cells, and KB epidermoid cancer cells. In addition, some functional chromones have the function of inducing apoptosis in A549 lung cancer cells.

Chromone is a good compound for structural modification and can synthesize a variety of pharmaceutically active molecules. Because of its ability to synthesize diverse structures, chromone is considered to be the leading compound in pharmacology.

However, in the chromone family, molecules having a skeleton of heteroatom-conjugated groups (eg, amine groups, halides, sulfides) are relatively rare, especially chromones having a sulfonyl substituent. However, sulfonamide compounds have valuable biological activity and are important substituents in the pharmaceutical field, such as sulfonamides.

Chromone is an oxygen-containing heterocyclic compound and has antioxidant function. Some chromones can regulate reactive oxygen species (ROS) and reactive nitrogen species (RNS). The mechanism of action of chromones with active oxygen species on cancer cells remains to be studied.

，化合物為2-萘基-1-基甲基-3-(甲苯-4-磺醯基) 苯并哌喃-4-酮。 The present disclosure provides a novel sulfonyl benzopyran-4-one compound, wherein the molecular formula of this compound has the following structure:

The compound is 2-naphthyl-1-ylmethyl-3-(toluene-4-sulfonyl) benzopiperan-4-one.

，其中該化合物為2-萘基-1-基甲基-3-(甲苯-4-磺醯基) 苯并哌喃-4-酮，且其具有選擇性殺死該口腔癌細胞之功效。 The present disclosure also provides a pharmaceutical composition for selectively killing an oral cancer cell, including: a sulfonyl benzopyran-4-one compound as an active ingredient; and a pharmaceutically acceptable carrier or salt , Where the molecular formula of the compound is as follows:

, Wherein the compound is 2-naphthyl-1-ylmethyl-3-(toluene-4-sulfonyl) benzopiperan-4-one, and it has the effect of selectively killing the oral cancer cells.

，其中該化合物為2-萘基-1-基甲基-3-(甲苯-4-磺醯基) 苯并哌喃-4-酮，且其具有選擇性殺死該口腔癌細胞之功效。 The disclosure also provides a use of a sulfonyl benzopyran-4-one compound for preparing a drug that selectively kills an oral cancer cell. The molecular formula of the compound is as follows:

, Wherein the compound is 2-naphthyl-1-ylmethyl-3-(toluene-4-sulfonyl) benzopiperan-4-one, and it has the effect of selectively killing the oral cancer cells.

，其中，該化合物為2-萘基-1-基甲基-3-(甲苯-4-磺醯基) 苯并哌喃-4-酮，且其具有於一癌細胞中造成氧化壓力之功效。 The present disclosure further provides the use of a sulfonyl benzopyran-4-one compound for the preparation of a cancer therapeutic drug using oxidative stress as a medium, wherein the molecular formula of the compound is as follows:

, Where the compound is 2-naphthyl-1-ylmethyl-3-(toluene-4-sulfonyl) benzopiperan-4-one, and it has the effect of causing oxidative stress in a cancer cell .

In order to make the above and other objects, features, and advantages of the present invention more comprehensible, preferred embodiments are described below in conjunction with the accompanying drawings, which are described in detail below.

In the present invention, by introducing a sulfonyl substituent into the chromone skeleton, a novel sulfonyl chromen-4-ones chromone compound is artificially synthesized, and Explore the effect of this chromone on cancer cells.

，其為2-萘基-1-基甲基-3-(甲苯-4-磺醯基) 苯并哌喃-4-酮(2-naphthalen-1-ylmethyl-3-(toluene-4-sulfonyl)chromen-4-one)。 According to the above, in the first embodiment of the present invention, a novel sulfonyl benzopyran-4-one compound is provided. The molecular formula of this compound is as follows:

, Which is 2-naphthalen-1-ylmethyl-3-(toluene-4-sulfonyl) benzopiperan-4-one (2-naphthalen-1-ylmethyl-3-(toluene-4-sulfonyl )chromen-4-one).

，     其中化合物為2-萘基-1-基甲基-3-(甲苯-4-磺醯基) 苯并哌喃-4-酮，且其具有選擇性殺死該口腔癌細胞之功效。 In addition, in the second embodiment of the present invention, a pharmaceutical composition for selectively killing oral cancer cells is provided. The above pharmaceutical composition may include, but is not limited to, monosulfonylbenzopiperan-4-one The compound is the active ingredient and a pharmaceutically acceptable carrier or salt, and the molecular formula of the above compound has the following structure:

, Wherein the compound is 2-naphthyl-1-ylmethyl-3-(toluene-4-sulfonyl) benzopiperan-4-one, and it has the effect of selectively killing the oral cancer cells.

In addition, the above sulfonyl benzopyran-4-one compound can selectively kill cancer cells, especially oral cancer cells. In other words, the compound is effective in killing cancer cells with little or no harm to normal cells.

In one embodiment, the compounds shown above may have the effect of causing oxidative stress in cancer cells, especially oral cancer cells. In another embodiment, the compounds shown above may have the effect of causing cancer cell cells to produce reactive oxygen species and/or superoxide, DNA damage and/or oxidative DNA damage, especially oral cancer cells. In yet another embodiment, the compound shown above may have the effect of apoptotic cancer cells, especially oral cancer cells. In some embodiments, examples of the oral cancer cells include gingival cancer cells and/or tongue cancer cells, but are not limited thereto.

The compound shown above can be artificially synthesized, and is not particularly limited.

In one embodiment, the above pharmaceutical composition can be used in a method of treating oral cancer, which can be used in combination with X-ray irradiation. In this particular embodiment, the dosage of the pharmaceutical composition for the combination therapy of the above-mentioned pharmaceutical composition and X-ray is reduced or the same as when the pharmaceutical composition is administered alone, and/or the combination therapy of the above-mentioned X-ray and the pharmaceutical composition The dosage of X-ray is reduced or the same as that when X-ray is administered alone. The combination therapy of the above-mentioned pharmaceutical composition and X-ray may have better efficacy of killing cancer cells than separate administration. Also, in this specific embodiment, in the combination therapy of the pharmaceutical composition and X-ray, the pharmaceutical composition and X-ray have a synergistic effect. Furthermore, in this specific embodiment, in the use in the preparation of the above-mentioned cancer treatment drug, the cancer includes oral cancer, but is not limited thereto. In this disclosure, the term "effective dose" refers to the lowest dose that can kill cancer cells or the lowest dose that can relieve the patient's disease.

In one embodiment, the X-ray irradiation dose may be about 2-20 Gy, such as about 8-15 Gy.

In a specific embodiment, when the combination of the pharmaceutical composition and X-ray is used for treatment, the dose of the pharmaceutical composition and the X-ray irradiation dose have a dose ratio, for example, 1:0.5~1:2 (μg/ml: Gy), 1:0.8~1:1.5 (μg/ml:Gy), for example, about 1:1.2 (μg/ml:Gy).

In addition, in the combination therapy of the aforementioned pharmaceutical composition and X-ray, the order of administration of the pharmaceutical composition and irradiation of X-ray is not particularly limited. For example, the pharmaceutical composition can be administered before X-ray irradiation, or alternatively, the pharmaceutical composition can be administered after X-ray irradiation, or the pharmaceutical composition and X-ray irradiation can be administered simultaneously get on.

In the third embodiment of the present disclosure, the use of a compound for preparing a drug for selectively killing an oral cancer cell is provided. The structural formula of the compound is as described above and has the effect of selectively killing oral cancer cells. The above drugs may further include pharmaceutically acceptable carriers and/or salts.

The drug for selectively killing oral cancer cells in the third embodiment has the same effect as the pharmaceutical composition for selectively killing oral cancer cells in the second embodiment. These include selective killing of cancer cells, oxidative stress in cancer cells, production of reactive oxygen species and/or superoxide in cancer cells, DNA damage and/or oxidative DNA damage, and apoptosis of cancer cells. In some embodiments, examples of the oral cancer cells include gingival cancer cells and/or tongue cancer cells, but are not limited thereto. The above-mentioned drugs can be used in combination with X-ray to kill oral cancer cells. Relevant instructions for the combined use are the same as in the pharmaceutical composition of the second embodiment.

In the fourth embodiment of the present disclosure, there is provided a compound for use in the preparation of a cancer therapeutic drug mediated by oxidative stress. The structural formula of this compound is as described above, and it has the effect of causing oxidative stress in cancer cells. The above drugs may further include pharmaceutically acceptable carriers and/or salts.

The fourth embodiment of the treatment of oxidative stress-mediated cancer treatment drugs has the same effect as the second embodiment of the pharmaceutical composition for selectively killing oral cancer cells. These include selective killing of cancer cells, oxidative stress in cancer cells, production of reactive oxygen species and/or superoxide in cancer cells, DNA damage and/or oxidative DNA damage, and apoptosis of cancer cells. In some embodiments, examples of the oral cancer cells include gingival cancer cells and/or tongue cancer cells, but are not limited thereto. The above-mentioned drugs can be used in combination with X-ray to kill oral cancer cells. Relevant instructions for the combined use are the same as in the pharmaceutical composition of the second embodiment.

In the present disclosure, the term "cancer drugs mediated by oxidative stress" refers to drugs that have the ability to increase the oxidative stress of cancer cells and thereby cause cancer cell death and/or decrease proliferation.

The above pharmaceutically acceptable carriers may include, but are not limited to, solvents, dispersion medium, coating, antibacterial and antifungal agents, and isotonic and absorption delaying agents, etc. Compatible. For different administration methods, the pharmaceutical composition can be formulated into a dosage form by a general method.

The above pharmaceutically acceptable salts may include, but are not limited to, salts including inorganic cations, for example, alkali metal salts such as sodium, potassium or amine salts, alkaline earth gold salts such as magnesium and calcium salts, containing divalent Or tetravalent cation salts, such as zinc, aluminum or zirconium salts. In addition, it can also be organic salts, such as dicyclohexylamine salts, methyl-D-glucosamine, amino acid salts, such as arginine, lysine, histidine, glutamine amide .

The pharmaceutical composition or drug of the present invention can be administered orally, parenterally or by means of an implanted reservoir. Non-oral may include subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intraarterial, intrasynovial, intrasternal (intrasternal), intrathecal, intralesional injection and perfusion techniques.

The form of oral ingredients may include, but is not limited to, tablets, capsules, emulsions, aqueous suspensions, dispersions, and solutions.

The pharmaceutical composition or medicine of the present invention can be administered to plants or animals. The above may include fish, amphibians, reptiles, birds, mammals, etc., but is not limited thereto. Examples of mammals may include, but are not limited to, cats, dogs, cows, horses, pigs, humans, and the like. In one embodiment, the pharmaceutical composition or drug can be administered to humans.

Preparation Example A. Materials and methods 1. Cell culture human oral cancer cell lines from gum cancer (Ca9-22) and tongue cancer (CAL 27) were purchased from Health Science Research Resources Bank (HSRRB) ) (Osaka, Japan) and the American Type Culture Collection (ATCC; Virginia, USA). Normal human gingival fibroblast beads (HGF-1) were purchased from ATCC. The cell culture medium is supplemented with 10% fetal bovine serum (FBS), penicillin (penicillin) 100 units/ml, streptomycin solution (streptomycin) 100 μg/ml in a conventional formula (Gibco, Grand Island, NY, USA). Gentamycin (gentamicin) 50 μg/ml, amphotericin B (amphotericin B) 2.5 μg/ml. The cells were cultured in an incubator at 37°C containing 5% CO ₂ and appropriate humidity.

2. Synthesis of sulfonylbenzopiperan-4-one At room temperature, 150 mg (1.1 mmol) of potassium carbonate (K ₂ CO ₃ ) was added to contain 0.5 mmol of 1-(2-hydroxyphenyl) 2-(Toluene-4-sulfonyl)ethanone (1-(2-hydroxyphenyl)-2-(toluene-4-sulfonyl) ethanone) in 10 ml of acetonitrile to form a reaction mixture. At room temperature, the reaction mixture was stirred for 5 minutes. Next, an acetonitrile solution (5 ml) containing 60 mg (0.5 mmol) of naphthalen-2-yl-acetic anhydride was added to the reaction mixture. In an extractor cabinet, stir for 2 hours. The solution was cooled to room temperature, and the solution was concentrated. The residue was diluted with water and extracted with 20 ml of dichloromethane (CH ₂ Cl ₂ ), a total of three times. The extracts (organic layers) obtained three times were combined, washed with brine, dried and filtered, and then the crude product was obtained under reduced pressure evaporation. Then purify with silica gel to obtain chromone CHW09 with sulfonyl substituent. This chromone compound is dissolved in dimethyl sulfoxide (DMSO) solution. The obtained chromone compound was subjected to nuclear magnetic analysis, and its high-resolution mass spectrometry (ESI, M ⁺ +1) theoretical value was C ₂₇ H ₂₁ O ₄ S 441.1161, and the measured value was 441.1166. NMR spectrum (400 MHz, CDCl ₃ ): δ 8.05 (dd, J = 1.6, 8.0 Hz, 1H), 7.91-7.88 (m, 2H), 7.67-7.63 (m, 1H), 7.50-7.37 (m , 6H), 7.30 (d, J = 8.0 Hz, 2H), 7.17-7.12 (m, 3H), 4.64 (d, J = 14.0 Hz, 1H), 4.19 (d, J = 13.6 Hz, 1H), 2.39 (s, 3H). Carbon nuclear magnetic resonance spectrum (100 MHz, CDCl ₃ ): δ 160.11, 153.26, 145.25, 140.60, 136.40, 133.54, 132.26, 131.03, 130.29, 129.87 (2x), 129.37, 128.75, 128.71, 128.03, 127.88 (2x), 127.28 , 126.99, 126.21, 125.29, 124.51, 124.17, 118.53, 117.03, 57.34, 21.58.

This compound was confirmed to be 2-naphthalen-1-ylmethyl-3-(toluene-4-sulfonyl) benzopiperan-4-one (2-Naphthalen-1-ylmethyl-3-(toluene-4- sulfonyl)chromen -4-one), which will be named CHW09 in the future for simplicity.

3. Statistical methods The data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA) and Tukey HSD Post Hoc Test were used to analyze group differences (JMP® 12 software). Data markers with non-repeating lowercase letters represent significant differences.

[Example 1] Cell survival rate of oral cancer cells and normal cells treated with CHW09

The cell survival rate was measured by MTS assay based on mitochondrial reductase activity (CellTiter 96® Aqueous One Solution, Promega, Madison, WI, USA). Ca9-22, CAL 27 and HGF-1 cells were seeded in 96-well plates at 4×10 ³ cells per well, and the culture medium per well was 0.1 ml. Cells were treated with control group (DMSO) or different concentrations of CHW09 for 24 hours. Next, the cells were reacted with MTS reagent at 37°C for 60 minutes, and then the absorption at 490 nm was measured with an ELISA reader (EZ Read 400 Research, BioChrom, Holliston, MA, USA). The results are shown in Figure 1.

Figure 1 shows the cell survival rate (%) of two oral cancer cells (Ca9-22 and CAL 27) treated with CHW09 and normal oral HGF-1 cells. As can be seen from Figure 1, after 24 hours of treatment with CHW09 at concentrations of 0, 10, 25, 50, 75, and 100 μg/ml, the cell viability of Ca9-22 and CAL 27 oral cancer cells decreased dose-dependently, respectively. Ca9-22 is 100, 70, 61, 44, 29, 19 (%); CAL 27 is 100, 88, 77, 60, 45, 34 (%). In contrast, the cell survival rate of HGF-1 cells decreased only slightly, which was 100, 86, 81, 73, 69, 56 (%). In the case where the processing time of 24 hours, CHW09 Ca9-22 of oral cancer cell IC ₅₀ values of 40 μg / ml. Therefore, 25 and 50 μg/ml lower and higher than the IC ₅₀ value were selected for subsequent experiments.

[Example 2] Cell cycle analysis

Cell cycle analysis was performed with DNA dye 7-aminoactinomycin (7AAD) (Biotium Inc., Hayward, CA). After the CaW-22 cells treated with CHW09 were fixed, they were cultured in 1 μg/ml 7AAD at 37°C for 30 minutes, and then the cells were suspended in phosphate buffered saline (PBS buffer). Afterwards, analysis was performed with a flow cytometer (Accuri C6). The result is shown in FIG. 2A, and the table of cell cycle statistical data is shown in FIG. 2B.

Figure 2 shows that the proportions of Ca9-22 cells treated with CHW09 at concentrations of 0, 25, and 50 μg/ml at G1 phase were 30, 40, and 43 (%), respectively. As can be seen from FIG. 2B, CHW09 slightly increased the cells in the G1 phase, but there was no statistically significant difference.

[Example 3] Apoptosis analysis by annexin V/7AAD

Apoptosis was assessed by Annexin V (Strong Biotech Corp, Taipei, Taiwan)/7AAD. Cells were seeded in 6-well dishes with 2×10 ⁵ cells per well and 2 ml of culture medium. Cells were treated with DMSO or CHW09 at 0, 25, 50 μg/ml for 24 hours. Place the cells in Annexin V-fluorescein isothiocyanate with a final concentration of 10 μg/ml and 7AAD with a final concentration of 1 μg/ml for 30 minutes. Flow cytometer (Accuri C6 ) For analysis. The results are shown in FIG. 3A, the horizontal axis is the annexin V signal intensity, and the vertical axis is the 7AAD signal intensity. Annexin V-positive and 7AAD-positive were judged to be positive for apoptosis (+), and the table of statistical data of positive apoptosis (+) is shown in FIG. 3B.

Figure 3B shows that the percentage of apoptosis (+) of Ca9-22 cells treated with 0, 25, 50 μg/ml CHW09 was 4, 8, and 8%, respectively. Show that the treatment of CHW09 can cause apoptosis.

[Example 4] Apoptosis of oral cancer Ca9-22 cells treated with CHW09 by caspase.

In order to further examine the degree of apoptosis of oral cancer Ca9-22 cells treated with CHW09, Western blotting was used to examine the cleaved apoptotic enzyme (cysteine aspartate specific protease) 3, 8, 9 And cleaved poly ADP-ribose polymerase (poly(ADP-ribose) polymerase (PARP), select β-actin (β-actin) as internal control (internal control). The samples were processed as follows. Ca9-22 cells were treated with 0, 25, 50 μg/ml CHW09 for 24 hours, and the apoptosis inhibitor Z-VAD-FMK (Selleckchem.com; Houston, TX) at a concentration of 25 μM, respectively Pretreatment was performed for 2 hours, and the free radical scavenger N-acetylcysteine (NAC) (Sigma; St. Louis, MO) at a concentration of 2 mM was subjected to pretreatment for 1 hour. After that, the treated cells are subjected to a general cell collection step, and proteins are extracted with Lysis buffer. The Western blot method is briefly described as follows. The extracted protein is analyzed by electrophoresis tank, and the protein in the electrophoresis film is transferred to the PVDF membrane, and then blocked with Tris buffer containing 5% skimmed milk powder overnight. . After that, the detection antigen is detected by the primary antibody recognizing the specific antigen and the secondary antibody corresponding to the primary antibody. The primary antibodies used and the conditions of use are shown in the table below. After that, color development was carried out with a color developing agent (ECL HRP). The results are shown in FIGS. 4A and 4B. [Table 1] Primary antibody Label Dilution ratio Cleaved caspase-8 (Asp391) (18C8) Cell signaling Technology 1:1000 Cleaved caspase-3 Cleaved caspase-3 (Asp175)(5A1E) Cell signaling Technology 1:1000 Cleaved caspase-9 (Asp330)(d2D4) Cell signaling Technology 1:1000 PARP Cleaved PARP (Asp214) (D64E10) Cell signaling Technology 1:1000 β-actin (clone AC-15) Sigma-Aldrich 1:5000

Figure 4A shows that the amount of lysed caspase 3, 8, 9 and lysed PARP protein of Ca9-22 cells treated with 25, 50 μg/ml CHW09 varies with the concentration of CHW09 treatment rise. However, the group pre-treated with the apoptosis inhibitor Z-VAD-FMK suppressed this phenomenon. As can be seen from FIG. 4A, the treatment of CHW09 can cause apoptosis. FIG. 4B shows that the amount of PARP-lysing protein of Ca9-22 cells treated with 25, 50 μg/ml CHW09 increased with the concentration of CHW09 treatment. However, the group pretreated with the free radical scavenger NAC suppresses this phenomenon.

[Example 5] Confirmation of intracellular reactive oxygen species (ROS)

ROS reactive dye dichlorofluorescein diacetate (2',7'-dichlorodihydrofluorescein diacetate, H ₂ DCF-DA) (Sigma-Aldrich, St. Louis, MQ, USA) can react with ROS and form detectable Fluorescent molecules measured. The drug-treated cells were placed in a final concentration of 100 nM H ₂ DCF-DA (dissolved in PBS) for 30 minutes. After collection and washing, the cells were resuspended in PBS, using Accuri C6 flow cytometer (Becton-Dickinson, Mansfield, MA, USA) and its built-in software to detect the fluorescence intensity, defined as ROS intensity. The results are shown in Figure 5A. The data is shown in Figure 5B. The definition of relative ROS positive condition is that in the control group without drug treatment, the fluorescence intensity of the cell population is divided into 50% for the left and right, and the fluorescence intensity is the reference. The fluorescence intensity is greater than the above reference value is defined as ROS Positive (ie, the area on the right).

As shown in FIG. 5B, the relative ROS positive staining ratio (mean%) of Ca9-22 cells treated with 0, 25, and 50 μg/ml CHW09 was 50%, 55%, and 75%, respectively. It shows that the ROS in the cells increases with the increase of the dose of CHW09. It is inferred that CHW09 can induce the cells to produce ROS.

[Example 6] Confirmation of intracellular mitochondrial membrane potential (MMP)

It has been reported that cationic cyanine dyes accumulate in cells in response to membrane potential, and there are reports that changes in membrane potential are related to apoptosis. Therefore, use the anthocyanin dyes DiOC ₂ (3)( 3-ethyl-2-[3-(3-ethyl-2(3H)-benzoxazolylidene)-1-propenyl]-benzoxazolium iodide) and CCCP (carbonyl cyanide 3 -chlorophenylhydrazone) kit (Invitrogen, San Diego, CA, USA) to confirm the intracellular mitochondrial membrane potential. The drug-treated cells were treated with DiOC ₂ (3) (dissolved in medium) at a final concentration of 20 nM for 30 minutes. After collection and washing, the cells were resuspended in PBS, using Accuri C6 flow cytometer (Becton-Dickinson, Mansfield, MA, USA) and its built-in software to detect the fluorescence intensity, defined as MMP intensity. The results are shown in Figure 6A. The data is shown in Figure 6B. The definition of relative MMP negative is defined as the fluorescence intensity value when the fluorescence intensity of the cell population is divided into 50% on the left and right sides in the control group without drug treatment, and the fluorescence intensity is less than the above reference value is defined as MMP Negative (ie, the area on the left).

As shown in FIG. 6B, the relative MMP negative staining ratios (mean%) of Ca9-22 cells treated with 0, 25, and 50 μg/ml CHW09 were 50%, 76%, and 87%, respectively. It showed that the MMP in the cells decreased with the increase of the dose of CHW09. It is inferred that CHW09 can increase the depolarization of mitochondria and cause oxidative stress in the cells.

[Example 7] Confirmation of intracellular mitochondrial superoxide (Mitochondrial superoxide, MitoSOX)

Confirmation of intracellular mitochondrial superoxide was performed using MitoSOX ^™ Red kit (Molecular Probes, Invitrogen, Eugene, OR, USA). The drug-treated cells were placed in MitoSOX with a final concentration of 5 μM and incubated for 30 minutes. After collection and washing, the cells were resuspended in PBS, using Accuri C6 flow cytometer (Becton-Dickinson, Mansfield, MA, USA) and its built-in software to detect the fluorescence intensity, defined as the MitoSOX intensity. The results are shown in Figure 7A, and the data are shown in Figure 7B. The relative definition of MitoSOX positive is that in the control group without drug treatment, the fluorescence intensity of the cell population is divided into 50% for each of the left and right sides. The fluorescence intensity is greater than the above reference value is defined as MitoSOX Positive (ie, the area on the right).

As shown in FIG. 7B, the relative MitoSOX positive staining ratios (mean%) of Ca9-22 cells treated with 0, 25, and 50 μg/ml CHW09 were 50%, 71%, and 70%, respectively. It was shown that the MitoSOX in the cells increased with the increase of the dose of CHW09. It is inferred that CHW09 can increase the accumulation of superoxide in the cells and cause oxidative stress in the cells. In addition, in the group pretreated with mitochondrial superoxide inhibitor mito-TEMPO (Cayman chemical, Ann Arbor, MI, USA), 20 μM for 1 hour, Ca9 treated with 0, 25, 50 μg/ml CHW09 The relative MitoSOX positive staining ratio (mean%) of -22 cells was 50%, 58% and 52%, respectively, which was significantly reduced compared with the untreated mito-TEMPO group. The above results further confirm that the treatment of CHW09 can cause the accumulation of mitochondrial superoxide in the cells, causing oxidative stress in the cells.

[Example 8] Confirmation of DNA damage in cells

γH2AX is a DNA double strand break marker, so detecting the content of γH2AX in a cell can reflect the degree of DNA damage in the cell. In order to confirm the effect of oxidative stress on cells caused by CHW09 treatment, confirmation of intracellular DNA damage was performed. Specifically, the drug-treated cells were fixed with 70% ethanol and fixed with BSA-T-PBS solution (PBS containing 1% bovine serum albumin (BSA) and 0.2% Triton X-100) ( Sigma) cleaning. After centrifugation, resuspend the cells at 4°C in a monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) containing 2 μl of anti-phospho-Histone (Histone) H2A.X (Ser 139) Incubate in 100 μl of BSA-T-PBS solution for 1 hour. After washing, the cells were resuspended in 100 μl of BSA-T-PBS solution containing 2 μl of Alexa Fluor 488-labeled secondary antibody (Jackson Laboratory, Bar Harbor, ME, USA) at room temperature and incubated for 30 minutes . After collecting the cells, resuspend the cells in PBS containing 5 μg/ml propidium iodide and analyze them with a flow cytometer (BD Accuri™ C6) to detect the fluorescence intensity, defined as the intensity of γH2AX. The result is shown in FIG. 8A, and the γH2AX positive data is shown in FIG. 8B. The definition of relative γH2AX positive is based on the fluorescent intensity value of the control group without drug treatment, when the fluorescence intensity of the cell population is divided into 50% on the left and right, and the fluorescence intensity is greater than the above reference value is defined as γH2AX positive (That is, the area on the right). In addition, the Western blot method was also used to detect the amount of γH2AX protein in cells treated with drugs. The Western blot method is carried out in the same manner as in Example 4 except that the primary antibody is replaced with phosphorylated-histone H2A.X (Ser139) (Santa Cruz Biotechnology), which will not be repeated here. The results are shown in Figure 8C.

As shown in FIG. 8B, the relative γH2AX positive staining ratios (mean%) of Ca9-22 cells treated with 0, 25, and 50 μg/ml CHW09 were 50%, 52%, and 55%, respectively. It shows that the content of phosphorylated γH2AX in cells increases with the increase of the dose of CHW09. It is inferred that CHW09 can increase the oxidative stress of cells and cause DNA damage. And the Western blot method shown in FIG. 8C also shows that the amount of phosphorylated γH2AX protein in Ca9-22 cells treated with 25, 50 μg/ml CHW09 increased.

[Example 9] Confirmation of DNA damage of oxidative stress in cells

When cells are exposed to reactive oxygen species (ROS), ROS attacks the guanine base in the DNA molecule, producing 8-oxo-2'-deoxyguanosine, 8-OxodG), so 8-OxodG can be used as an indicator of DNA damage due to ROS.

Using the kit for detecting 8-OxodG OxyDAN analysis kit (no. 500095; EMD Millipore, Darmstadt, Germany), the cells were stained according to the instructions attached to the kit, and flow cytometry (BD Accuri™ C6) Analysis and detection of fluorescence intensity, defined as 8-OxodG intensity. The results are shown in Figure 9A, and the data are shown in Figure 9B. The relative definition of 8-OxodG positive is that in the control group without drug addition, the fluorescence intensity of the cell population is divided into 50% for the left and right, and the fluorescence intensity is the reference, and the fluorescence intensity is greater than the above definition 8-OxodG positive (ie, the area on the right).

As shown in FIG. 9B, the relative 8-OxodG positive staining ratio (mean%) of Ca9-22 cells treated with 0, 25, and 50 μg/ml CHW09 were 50%, 52%, and 62%, respectively. It showed that the content of 8-OxodG in cells increased with the increase of the dose of CHW09. It is inferred that CHW09 can increase the oxidative stress of cells and cause DNA damage.

[Example 10] Cell survival rate of drug combined with X-ray

In recent years, radiotherapy (RT) has become one of the important methods for treating malignant tumors. Because radiation therapy can be located in the area of the tumor, the combination of drugs and radiation therapy can achieve low-dose and high-efficiency treatment. After Ca9-22 and HGF-1 cells were irradiated or not irradiated with X-ray (12 Gy) (6 MV photon linear accelerator (Elekta Axesse; Stockholm, Sweden)), they were treated with CHW09 at a final concentration of 10 μg/ml After 24 hours or 24 hours without treatment with CHW09, the cell survival rate was measured using the same method as in Example 1, and the results are shown in FIG. 10.

As shown in Figure 10, the cell survival rate of Ca9-22 cells treated with CHW09 alone at a final concentration of 10 μg/ml was 87.25±1.14%; the survival rate of Ca9-22 cells irradiated with X-ray alone was 86.54±0.33% . In contrast, the survival rate of Ca9-22 cells treated with CHW09 and irradiated with X-ray was 73.48±0.27%. From this result, it can be seen that the combined use of CHW09 and X-ray has a synergistic effect. In addition, the survival rate of HGF-1 cells treated with CHW09 and irradiated with X-ray was 84.97±3.08%, which was significantly different from the survival rate of Ca9-22 cells. According to the above experiments, CHW09 and X-ray can selectively kill oral cancer cells.

From the above results, it can be seen that treatment with CHW09 induces cells to produce ROS, reduce MMP, increase mitochondrial superoxide and other oxidative stress, and cause DNA damage and oxidative DNA damage, which ultimately leads to apoptosis. Compared with normal cells, CHW09 will cause a lower survival rate of oral cancer cells, that is, CHW09 can selectively kill oral cancer cells. In addition, the combined use of CHW09 and X-ray has a synergistic effect, which can effectively kill oral cancer cells at a lower dose than when used alone.

Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this skill can make some modifications and retouching without departing from the spirit and scope of the present invention. Therefore, the present invention The scope of protection shall be deemed as defined by the scope of the attached patent application.

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Figure 1 shows the cell survival rate of oral cancer cells and normal cells treated with CHW09. Ca9-22 and CAL 27 oral cancer cells and HGF-1 normal oral cells were treated with CHW09 (0, 10, 25, 50, 75 and 100 μg/ml) for 24 hours. MTS assays were performed to determine cell viability. The data is the mean ± SD (n = 3). Data markers with non-repeating lowercase letters represent significant differences. Figure 2A shows the changes in the cycle of Ca9-22 oral cancer cells treated with CHW09. Cells were treated with CHW09 (0, 25, and 50 μg/ml) for 24 hours. Next, the 7-aminoactinomycin D (7AAD) intensity of the Ca9-22 cells treated with CHW09 was calculated using a flow cytometer, the horizontal axis is the signal intensity, and the vertical axis is the number of cells. Figure 2B shows the statistics of the proportion of cells divided by the cell cycle in Figure 2A. The data is the mean ± SD (n = 3). Data markers with non-repeating lowercase letters represent significant differences. Figure 3A shows the change of apoptosis of Ca9-22 oral cancer cells treated with CHW09. Cells were treated with CHW09 (0, 25, and 50 μg/ml) for 24 hours. Next, annexin V/7AAD of CaW-22 cells treated with CHW09 was calculated using a flow cytometer. Annexin V(+)/7AAD(+) is calculated as positive for apoptosis (+)%, the horizontal axis is annexin V (annexin V) signal intensity, and the vertical axis is 7AAD signal intensity. Figure 3B Statistics of positive (+) (%) apoptosis. The data is the mean ± SD (n = 3). Data markers with non-repeating lowercase letters represent significant differences. Figure 4A shows the expression of apoptotic proteins (lysed apoptotic enzymes 3, 8, 9 and PARP) of Ca9-22 oral cancer cells treated with CHW09 or pre-treated with Z-VAD-FMK before CHW09 treatment Analysis of the Western blot method. Β-actin was used as an internal control. Figure 4B shows Western blot analysis of the expression of apoptotic proteins (lysed PARP) in Ca9-22 oral cancer cells pretreated with CHW09 or pretreated with NAC before CHW09 treatment. Β-actin was used as an internal control. Figure 5A shows the ROS changes of Ca9-22 oral cancer cells treated with CHW09. Cells were treated with CHW09 (0, 25, 50 μg/ml) for 24 hours. Then use flow cytometry to detect the fluorescence intensity of ROS. The horizontal axis is the signal strength, and the vertical axis is the number of cells. Figure 5B shows the statistics of ROS positive (+) intensity (%) of Figure 5A. The data is the mean ± SD (n = 3). Data markers with non-repeating lowercase letters represent significant differences. Figure 6A shows the MMP changes of Ca9-22 oral cancer cells treated with CHW09. Cells were treated with CHW09 (0, 25, 50 μg/ml) for 24 hours. Then use flow cytometry to detect the fluorescence intensity of MMP. The horizontal axis is the signal strength, and the vertical axis is the number of cells. Figure 6B shows the statistics of the MMP negative (-) intensity (%) of Figure 6A. The data is the mean ± SD (n = 3). Data markers with non-repeating lowercase letters represent significant differences. Figure 7A shows the changes in mitochondrial superoxide (MitoSOX) production of Ca9-22 cells treated with CHW09. Cells were treated with CHW09 (0, 25, 50 μg/ml) for 24 hours. Then use flow cytometry to detect the fluorescence intensity of ROS. The figure shows the pattern of Ca9-22 cells with or without mito-TEMPO treatment. The horizontal axis is the signal strength, and the vertical axis is the number of cells. Figure 7B shows the mitochondrial superoxide positive (+) intensity (%) statistics of Figure 7A. The data is the mean ± SD (n = 3). Figure 8A shows the change of γH2AX in Ca9-22 oral cancer cells treated with CHW09. Cells were treated with CHW09 (0, 25, 50 μg/ml) for 24 hours. Then use a flow cytometer to detect the fluorescence intensity of γH2AX. The horizontal axis is the signal strength, and the vertical axis is the number of cells. Figure 8B shows the statistics of the positive (+) intensity (%) of γH2AX in Figure 8A. The data is the mean ± SD (n = 3). Data markers with non-repeating lowercase letters represent significant differences. Figure 8C shows Western blot analysis of phosphorylated γH2AX protein in Ca9-22 oral cancer cells treated with CHW09. Β-actin was used as an internal control. Figure 9A shows the 8-OxodG changes of Ca9-22 oral cancer cells treated with CHW09. Cells were treated with CHW09 (0, 25, 50 μg/ml) for 24 hours. Then use flow cytometry to detect the fluorescence intensity of 8-OxodG. The horizontal axis is the signal strength, and the vertical axis is the number of cells. Figure 9B shows the statistics of the 8-OxodG positive (+) intensity (%) of Figure 9A. The data is the mean ± SD (n = 3). Data markers with non-repeating lowercase letters represent significant differences. Figure 10 shows the cell survival rate of oral cancer cells and normal cells treated with CHW09 and/or X-ray. ChW09 (10 μg/ml) and/or X-ray (12 Gy) were used to treat Ca9-22 and CAL 27 oral cancer cells and HGF-1 normal oral cells for 24 hours. MTS assays were performed to determine cell viability. The data is the mean ± SD (n = 3). Data markers with non-repeating lowercase letters represent significant differences.

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Claims (9)

A novel sulfonyl benzopyran-4-one compound, wherein the molecular formula of the compound is as follows:

The compound is 2-naphthyl-1-ylmethyl-3-(toluene-4-sulfonyl)benzopiperan-4-one. A pharmaceutical composition for selectively killing an oral cancer cell, comprising: a sulfonyl benzopyran-4-one compound as an active ingredient; and a pharmaceutically acceptable carrier or salt, wherein the compound The molecular formula is as follows:

, Wherein the compound is 2-naphthyl-1-ylmethyl-3-(toluene-4-sulfonyl) benzopiperan-4-one, and it has the effect of selectively killing the oral cancer cells. The pharmaceutical composition for selectively killing an oral cancer cell as described in item 2 of the patent application scope, wherein the oral cancer cell includes a gingival cancer cell and/or a tongue cancer cell. The pharmaceutical composition for selectively killing an oral cancer cell as described in item 2 of the patent application scope, wherein the pharmaceutical combination for selectively killing an oral cancer cell The compound is used in a method for treating oral cancer, and in this method, the pharmaceutical composition is irradiated with X-ray and used to selectively kill oral cancer cells. The use of a sulfonyl benzopyran-4-one compound for the preparation of a drug that selectively kills an oral cancer cell, wherein the molecular formula of the compound is as follows:

, Wherein the compound is 2-naphthyl-1-ylmethyl-3-(toluene-4-sulfonyl) benzopiperan-4-one, and it has the effect of selectively killing the oral cancer cells. The use of the compound as described in item 5 of the patent application for the preparation of a drug that selectively kills an oral cancer cell, wherein the oral cancer cell includes a gingival cancer cell and/or a tongue cancer cell. The use of a sulfonyl benzopiperan-4-one compound for the preparation of oral cancer treatment drugs using oxidative stress as a medium, wherein the molecular formula of the compound is as follows:

, Wherein the compound is 2-naphthyl-1-ylmethyl-3-(toluene-4-sulfonyl) benzopiperan-4-one, and it has the effect of causing oxidative stress in a cancer cell. The use of the compound as described in item 7 of the patent application for the preparation of oral cancer treatment drugs using oxidative stress as a medium, wherein the compound has the effect of selectively killing an oral cancer cell. The use of the compound as described in item 8 of the patent application for the preparation of an oral cancer treatment drug using oxidative stress as a medium, wherein the oral cancer includes a gum cancer and/or a tongue cancer.

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