An artificial H + / CI - Preparation of ion channels

Abstract

The application provides an artificial H + / Cl ‑ The preparation of the ion channel, the compound has a molecular skeleton of resorcinol and is modified by different side chains, and a general structure is as follows: the compound involved in the application has good proton and chloride ion transmission activity, and shows good anti-tumor activity and selectivity, and has the potential to be developed into a new anti-tumor drug. The synthetic route of the application is green, simple, efficient, and raw materials are easy to obtain, and can be used for the synthesis of similar compounds.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a preparation of a visible light responsive artificial H+ / Cl- ion channel with specific anti-tumor activity. BACKGROUND

[0002] Cancer is one of the diseases that causes the most deaths worldwide. According to the World Health Organization (WHO) report, cancer is the second leading cause of death worldwide, with nearly 10 million deaths per year. Lung cancer, prostate cancer, colorectal cancer, gastric cancer and liver cancer are the most common types of cancer in men, while breast cancer, colorectal cancer, lung cancer, cervical cancer and thyroid cancer are the most common in women. Obviously, the burden of cancer brings huge physical, emotional and economic pressure to individuals, families, communities and health systems worldwide. Cancer remains a major problem worldwide.

[0003] Although in the past few decades, monoclonal antibody therapy, immunotherapy and gene therapy drugs are rapidly developing, due to the high cost of treatment, the current tumor treatment strategy is still dominated by chemotherapy. However, traditional chemotherapy drugs (cisplatin, daunorubicin, 5-fluorouracil, etc.) have poor selectivity for normal cells and tumor cells, often causing adverse reactions such as nausea, vomiting, hair loss, etc., which seriously affect the treatment effect. In addition, due to immunosuppression, the risk of infection increases dramatically. Therefore, it is an urgent need to find a highly specific anticancer drug.

[0004] The present application aims to solve the above problems, based on the differences in the microenvironment of tumor cells and normal cells and the characteristics of the response to visible light stimulation, a class of artificial H + / Cl - ion transporters with high selectivity, low drug resistance and low toxicity and side effects is developed, and it is applied to the treatment of specific anti-tumor.

[0005] SUMMARY

[0006] In order to overcome the shortcomings of the prior art, the present application provides a preparation of a visible light responsive artificial H + / Cl - ion channel with specific anti-tumor activity, which uses resorcinol as the molecular skeleton and modifies it with different side chains.

[0007] The technical solution adopted by the present application mainly includes three parts: first, the synthesis of artificial H + / Cl - ion channel, second, the detection of the transmembrane transport activity and ion selectivity of the target compound, and third, the visible light responsive prodrug modification of the high transport activity target compound and its application in specific anticancer research.

[0008] The visible light activated artificial H + / Cl - The ion channel structure is as follows:

[0009]

[0010] After visible light stimulation, the protecting group is removed to cause the inversion of the configuration of the amide bond, and the channel molecule restores the self-assembly function to transport H + / Cl - ions.

[0011] The synthesis route of the target compound is as follows:

[0012]

[0013] The specific synthesis process is as follows: (1) the channel compound is synthesized by connecting different side chains with resorcinol as a molecular skeleton; (2) the high-activity transmembrane transport molecule is modified by visible light response.

[0014] Further, the use of the compound obtained by the preparation method in visible light anticancer treatment.

[0015] A pharmaceutical composition comprising the compound obtained by the preparation method and a pharmaceutical excipient.

[0016] The beneficial effects of the present application are:

[0017] 1. The synthesis route of the present application is green, simple, efficient, and the raw materials are easy to obtain, and can be used for the synthesis of similar compounds.

[0018] 2. The compound involved in the present application has good proton and chloride ion transport activity; specifically, the pH-sensitive HPTS vesicle fluorescence experiment based on the compound proves that the compound has transmembrane transport activity; the SPQ vesicle fluorescence experiment based on the chloride ion sensitivity shows that the compound has excellent chloride ion transmembrane transport activity; the single-molecule channel current experiment proves that the compound has the ability to quickly transport hydrogen ions and chloride ions; the nuclear magnetic resonance hydrogen spectrum and HPTS vesicle fluorescence experiment prove that the visible light modified precursor compound can quickly decompose and restore the channel activity.

[0019] 3、In vitro anti-tumor experiments show that the compounds exhibit good anti-tumor activity and selectivity, and have the potential to develop into new anti-tumor drugs. Specifically, the prepared compounds can induce human colon cancer cells (HCT-116) to undergo apoptosis and have little cytotoxicity to normal intestinal cells (HIEC-6); the prepared compounds can cause depolarization of the mitochondrial membrane potential and lead to an increase in the content of ROS in cancer cells; the prepared compounds can lead to down-regulation of typical anti-apoptotic proteins MCL1 and BCL2 and cause tumor cell death by activating the Caspase 9 signaling pathway; the prepared compounds can change the pH of lysosomes and thereby interfere with lysosomal autophagy. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Schematic diagram of the results of the study on the transmembrane transport activity of the channel molecule;

[0021] Figure 2 Schematic diagram of the results of the transmembrane transport mechanism of the channel molecule;

[0022] Figure 3 a to c in FIG. 3 are schematic diagrams of the results of single-molecule channel current, Figure 3 a in FIG. 3 is a single-channel current trace; b in FIG. 3 is a current-voltage (I-V) curve of C4F; c in FIG. 3 is a P H + / P Cl - ion selectivity ratio of C4F-L;

[0023] Figure 4 Schematic diagram of the results of the cleavage of C4F-L under visible light;

[0024] Figure 5 Schematic diagram of the results of the visible light activation of C4F-L;

[0025] Figure 6 Schematic diagram of the results of the inhibition rate of C4F-L on HCT-116 cells;

[0026] Figure 7 Schematic diagram of the results of the inhibition rate of C4F-L on HIEC-6 cells;

[0027] Figure 8 Schematic diagram of the results of the induction of HCT-116 cell apoptosis by C4-L after visible light activation;

[0028] Figure 9 Schematic diagram of the results of the depolarization of the mitochondrial membrane potential caused by C4-L after visible light activation;

[0029] Figure 10 Schematic diagram of the results of the change in the content of intracellular ROS caused by C4-L after visible light activation;

[0030] Figure 11 Figure 2 shows the results of immunoblot analysis of C4F-L on HCT-116 cells;

[0031] Figure 12 Figure 4 shows the results of lysosomal pH elevation and autophagy-related protein upregulation. DETAILED DESCRIPTION

[0032] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the present application is further described below in combination with specific embodiments, but the following embodiments are only preferred embodiments of the present application, not all. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application. In the following examples, the experimental methods are conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0033] Example 1:

[0034] Preparation of a visible light-responsive artificial H+ / CI- ion channel with specific anti-tumor activity, as shown below:

[0035]

[0036] Compound synthesis method:

[0037]

[0038] C2H. Yield: 0.5 g, yield: 87%. 1 H NMR (600 MHz, DMSO-d6) δ 13.02 (s, 2H), 8.46 (t, J = 5.3 Hz, 2H), 8.31 (s, 1H), 6.30 (s, 1H), 3.35-3.29 (m, 4H), 1.15 (t, J = 7.2 Hz, 6H). 13 C NMR (151 MHz, DMSO-d6) δ 168.74, 164.72, 129.25, 108.10, 103.83, 79.56, 79.34, 79.12, 72.74, 67.48, 60.76, 34.28, 25.58, 15.06. MS-ESI: calculated for [M+H] + (C 12 H 16 N2O4): m / z 253.1188, found: m / z 253.1185.

[0039] N2O4): m / z 253.1188, found: m / z 253.1185.

[0040] 129.75, 108.53, 103.84, 41.11, 22.76, 11.89. MS-ESI: calculated for [M+H] + (C 14 H 20 N2O4): m / z 303.1321, found: m / z 303.1311.

[0041]

[0042] calculated for [M+H] + (C 16 H 24 N2O4): m / z 309.1814, found: m / z 309.1804.

[0043]

[0044] (C 12 H 10 F6N2O4): m / z 359.0472, found: m / z 309.0466.

[0045]

[0046] 461.0559, found: m / z 461.0560.

[0047]

[0048]

[0049]

[0050] 12.63 (s, 1H), 8.48 (s, 1H), 8.34 (d, J = 1.8 Hz, 1H), 7.82 (dd, J = 8.2, 1.8 Hz, 2H), 7.48 (d, J = 8.3 Hz, 1H), 7.02 (s, 1H), 6.52 (s, 1H), 5.60 (s, 2H), 4.18 (dt, J = 15.4, 7.9 Hz, 4H). 13 C NMR (151 MHz, CDC13) δ 169.45, 166.61, 164.55, 160.52, 148.05, 137.28, 132.03, 131.02, 129.09, 128.73, 123.55, 112.31, 108.18, 101.86, 68.15. MS-ESI: calculated for [M+K]+ (C 23 H 14 BrF 14 N3O6):m / z 811.9479,found:m / z 811.9484.

[0051]

[0052] 132.10,131.08,129.10,128.76,123.60,112.37,108.25,101.89,68.18.MS-ESI:calculated for [M+K] + (C 27 H 14 BrF 22 N3O6):m / z 1011.9352,found:m / z 1011.9365.

[0053] Experimental Example 1

[0054] Detection of transmembrane transport activity of compounds

[0055] Twenty-five milligrams of egg yolk L-a-phosphatidylcholine (EYPC, Avanti Polar Lipids, USA) was dissolved in 1.0 mL of CHCl3. CHCl3was removed under reduced pressure at 35 °C to give a thin film, which was hydrated under vacuum at 25 °C overnight. The thin film was hydrated with a pH-sensitive HPTS dye (1.0 mM) in a HEPES buffer solution (1.0 mL, 10 mM HEPES, 100 mM NaCl, pH = 7.0) in a orbital shaker at 25 °C for 1 h to give a milky suspension. The mixture was then subjected to 10 freeze-thaw cycles: freezing in liquid nitrogen for 60 s and heating at 55 °C for 120 s. The vesicle suspension was extruded 21 times through polycarbonate membranes (0.1 pm) to produce a homogeneous suspension of LUVs with HPTS encapsulated inside. The same HEPES buffer solution (300 mL, without HPTS) was used to dialyze the suspension of LUVs against the HEPES buffer solution at gentle stirring (300 r / min, 4 °C) to remove the unencapsulated HPTS to produce LUVs with a lipid concentration of 6.5 mM.

[0056] A LUV stock solution (30 μL) containing HPTS was added to a HEPES buffer solution (1.95 mL, 10 mM HEPES, 100 mM NaCl, pH 8.0) to create a pH gradient for ion transport studies. The channel's DMSO solution was then injected into the suspension with gentle stirring. Immediately after channel addition, HPTS emission was recorded at 510 nm using a fluorescence spectrophotometer (Hitachi, model F-7100, Japan), with excitation at 403 and 403 nm for 300 seconds. At 300 seconds, 20 μL of Triton X-100 (20% v / v) was added to induce the maximum change in fluorescent dye emission. The final transport trajectory was recorded as I... 460 / I 403 The ratio value is obtained, and after adding triton using equation (1), it is based on I. 460 / I 403 The ratio values ​​are normalized.

[0057] I f =[(I t -I0) / (I1-I0)] (1)

[0058] Among them, I f = Fractional emission intensity, I t = Fluorescence intensity at time t, I1 = Fluorescence intensity after adding Triton X-100, I0 = Initial fluorescence intensity.

[0059] Using Hill's equation (2) to fit a partial ion transport activity R to the channel concentration, we obtain EC 50 Value and Hill coefficient n.

[0060] R = 1 / (1+(EC) 50 / [aisle]) n (2)

[0061] Depend on Figure 1 It can be seen that among the above 7 compounds, C4F and C6F are the compounds with the strongest transmembrane transport activity.

[0062] Further comparison of Cl was performed in the HPTS assay using FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, a highly active proton transporter). - and H + or OH - The ion transport rate between them. And combined with valamicin (VA, a highly active K+). + Selective transporter protein (CLP) experiments to compare Cl - and OH - The ion transport rate between them.

[0063] Depend onFigure 2 As shown, C4F (0.35 μM) exhibited almost the same ion transport activity in the presence (65%) and absence (62%) of FCCP (1.0 μM), indicating that there was no synergistic effect when the two were used together, which indicated that H + or OH - transported faster than Cl - . Similarly, VA and the channel did not exhibit a synergistic effect, indicating that Cl - transported faster than OH - . From the results of both experiments, it can be concluded that H + / Cl - symport was the main transport mechanism and H + transported faster than Cl - , and Cl - transported faster than OH - .

[0064] Ion transport based on SPQ vesicle fluorescence experiments

[0065] Twenty-five 0 mg of egg yolk L-a-phosphatidylcholine (EYPC, Avanti Polar Lipids, USA) was dissolved in 1.0 mL of CHCI3. CHCI3was removed under reduced pressure at 35 °C to give a thin film, which was hydrated at 25 °C under high vacuum overnight. The thin film was hydrated with NaNO3solution (1.5 mL, 200 mM) containing the chloride-sensitive dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) (0.5 mM) in a orbital shaker at 25 °C for 1 h to give a milky suspension. The mixture was then subjected to 10 freeze-thaw cycles: freezing in liquid nitrogen for 60 s and heating at 55 °C for 120 s. The vesicle suspension was extruded 21 times through a polycarbonate membrane (0.1 μm) to produce a uniform suspension of LUVs with SPQ encapsulated inside. The suspension of LUVs was produced by dialysis against the same NaNO3buffer solution (200 mM, without SPQ) using a membrane tube (MWCO = 10,000) to remove unencapsulated SPQ, resulting in a lipid LUV concentration of 6.5 mM.

[0066] LUV suspensions containing SPQ (30 μL) were added to NaCl solutions (1.95 mL, 200 mM) to create an extravesicular chloride gradient. Different concentrations of DMSO- channel solutions were then injected into the suspension under gentle stirring. Immediately after channel addition, the emission of SPQ was recorded at 430 nm using a fluorescence spectrophotometer (Hitachi, model F-7100, Japan) with excitation at 360 nm for 300 s, and then the chloride gradient was completely destroyed using Triton X-100 (20 μL added, 20% v / v). The final transport traces were obtained by normalizing the fluorescence intensities using equation (3).

[0067] I f = (I t - I1) / (I0-I1) (3)

[0068] The experimental results are shown in Table 1, where we observed a rapid quenching of the fluorescence intensity of SPQ with a concentration-dependent trend. The corresponding EC values for C4F and C6F were as low as 8.2 and 4.5 nM, respectively. 50 The experiments unambiguously confirmed that C4F and C6F have a very high efficiency in transporting chloride ions across the membrane.

[0069] Table 1. Chloride ion transport activity across the membrane of channel molecules

[0070] EC 50 (CI - )(nM)]]> C4F 8.23±0.50 C6F 4.52±0.16

[0071] Single-molecule channel current experiments demonstrate the ability of the compounds to form channels and rapidly transport hydrogen and chloride ions

[0072] To measure the chloride conductance (γ), a chloroform solution of diPhyPC (10 mg / ml, 40 μL) was evaporated under N2to form a thin film, which was then redissolved in 16 μL of n-decane. 0.5 μL of the n-decane solution containing the lipid was injected into the experimental chamber, which was then filled with 1.95 mL of 200 mM NaCl solution. Warner Instruments, Hamden, CT) and the n-decane was removed using N2. In the experiment, the cis chamber was filled with KCl aqueous solution (1.0 M, 1.0 mL) and the Delrin cup (trans chamber) was filled with the same solution. An Ag-AgCl electrode was inserted into both solutions and the cis chamber was grounded. A planar lipid bilayer was formed by painting 0.5 μL of the lipid-containing n-decane solution around the pre-treated well of n-decane. A planar lipid bilayer was indicated by a capacitance value in the range of 80-120 pF. The sample in DMSO (1.0 μL) was added to the cis compartment to achieve a final concentration of about 1 μM. The single-channel currents were then measured using a Warner BC-535D bilayer clamped amplifier, collected by PatchMaster (HEKA) at a sampling interval of 5 kHz and filtered using an 8-pole Bessel filter at 1 kHz (HEKA). The data were analyzed by FitMaster (HEKA) and a digital filter at 100 Hz. The current trace versus voltage plot was plotted to give the chloride conductance (γ).

[0073] The ion transport selectivity of H+versus Cl- was measured: Monoolein (15.0 mg) and cholesterol (15 mg) were dissolved in 0.5 ml of chloroform / methanol (2 / 1, v / v) to make a stock solution of lipids. 50 μL of the lipid-containing solution was added to the sample vial along with the channel solution in methanol. The solution was evaporated in N2to form a thin film, which was re-dissolved in 50 μL of n-decane to yield a working solution with a channel to GMO molar ratio of 1 / 106. 0.5 μL of the lipid-containing n-decane solution was injected into the well of the Delrin cup (Warner Instruments, Hamden, CT) and the n-decane was removed using N2. In the experiment, the cis chamber was filled with KCl aqueous solution (1.0 M, 1.0 mL) and the Delrin cup (trans chamber) was filled with the same solution. An Ag-AgCl electrode was inserted into both solutions and the cis chamber was grounded. A planar lipid bilayer was formed by painting 0.5 μL of the lipid-containing n-decane solution around the pre-treated well of n-decane. A planar lipid bilayer was indicated by a capacitance value in the range of 80-120 pF. The sample in DMSO (1.0 μL) was added to the cis compartment to achieve a final concentration of about 1 μM. The single-channel currents were then measured using a Warner BC-535D bilayer clamped amplifier, collected by PatchMaster (HEKA) at a sampling interval of 5 kHz and filtered using an 8-pole Bessel filter at 1 kHz (HEKA). The data were analyzed by FitMaster (HEKA) and a digital filter at 100 Hz. The current trace versus voltage plot was plotted to give the chloride conductance (γ). The ion transport selectivity of H+versus Cl- was measured: Monoolein (15.0 mg) and cholesterol (15 mg) were dissolved in 0.5 ml of chloroform / methanol (2 / 1, v / v) to make a stock solution of lipids. 50 μL of the lipid-containing solution was added to the sample vial along with the channel solution in methanol. The solution was evaporated in N2to form a thin film, which was re-dissolved in 50 μL of n-decane to yield a working solution with a channel to GMO molar ratio of 1 / 106. 0.5 μL of the lipid-containing n-decane solution was injected into the well of the Delrin cup (Warner Instruments, Hamden, CT) and the n-decane was removed using N2. In the experiment, the cis chamber was filled with KCl aqueous solution (1.0 M, 1.0 mL) and the Delrin cup (trans chamber) was filled with the same solution. An Ag-AgCl electrode was inserted into both solutions and the cis chamber was grounded. A planar lipid bilayer was formed by painting 0.5 μL of the lipid-containing n-decane solution around the pre-treated well of n-decane. A planar lipid bilayer was indicated by a capacitance value in the range of 80-120 pF. The sample in DMSO (1.0 μL) was added to the cis compartment to achieve a final concentration of about 1 μM. The single-channel currents were then measured using a Warner BC-535D bilayer clamped amplifier, collected by PatchMaster (HEKA) at a sampling interval of 5 kHz and filtered using an 8-pole Bessel filter at 1 kHz (HEKA). The data were analyzed by FitMaster (HEKA) and a digital filter at 100 Hz. The current trace versus voltage plot was plotted to give the chloride conductance (γ). H + / P Cl - ), can be obtained by fitting the I-V curve using the following simplified Goldman-Hodgkin-Katz equation (4).

[0074] ε rev +17.8 = RT / F x ln{(P H + [H + ] trans +P Cl - [Cl - ] cis) / (P H + [H + ] cis +P Cl - [Cl - ] trans )} (3)

[0075] Where ε rev This is the reverse membrane potential; 17.8 mV is the Nernst potential corresponding to the proton gradient from 0.025 M to 0.05 M. R is the universal gas constant (8.314 J K). -1 mol -1 T is the Kelvin temperature (298 K); F is the Faraday constant (96485 C mol). -1 P is the permeability of the ion channel.

[0076] like Figure 3 The C4F shown mediates Cl through a channel mechanism rather than a carrier mechanism. - Transmembrane transport. The Cl- of C4F was determined by linearly fitting the current-voltage (IV) plot. - Conductivity (γ) Cl - The value was 16.8 ± 0.3 ps. Furthermore, the H of C4F... + / Cl - Transmission selectivity (P) H + / P Cl - The calculated value is 6.0, further confirming H. + The transmission speed is higher than Cl - quick.

[0077] based on 1 ¹H NMR confirmed the ability of visible light-modified molecules to transform into active channel molecules under visible light irradiation.

[0078] A C4F-L (8mM) solution of deuterated dimethyl sulfoxide (DMSO-d6) was irradiated with a 24W LED (λem = 415nm), and the readings were recorded at appropriate time intervals. 1 H NMR spectrum.

[0079] The results are as follows Figure 4 As shown, the phenolic proton H in C4F-L a (12.58ppm), amide proton H b (8.54ppm) and H c (9.24 ppm) and methylene proton H d The signal corresponding to (5.57 ppm) disappears, and at the same time, the signal corresponding to the phenolic proton immediately appears.a' (12.31 ppm) and amide proton H b' (8.95ppm) clearly demonstrates the effective photostimulation release of C4F.

[0080] HPTS experiments demonstrated that visible light-modified molecules regain their ion transport capabilities after visible light irradiation.

[0081] DMSO solutions containing C4F-L (0.5 μM) and C6F-L (0.35 μM) were irradiated with an LED lamp at 20-second intervals, and their transmembrane transport activities were tested. The results are as follows: Figure 5 As shown, C4F-L and C6F-L can restore 90% of their transmembrane transport activity with just 2 minutes of visible light stimulation at low concentrations.

[0082] Experimental Example 2

[0083] Anticancer activity test

[0084] HCT-116 cells were seeded into 96-well plates, with a cell density of 1 × 10⁶ cells per well in 100 μL of DMEM. 4 Cells were used. The channel was dissolved in DMSO and diluted with serum-free medium. Cells treated with 0.5% DMSO served as a negative control. Once the HCT-116 cell density reached 50-60%, the medium in the 96-well plates was replaced with serum-free medium containing gradient concentrations of the channel reagent. After 24 hours of incubation, the medium was replaced with MTT solution. After 4 hours, the medium in the 96-well plates was removed, and 150 μL of DMSO was added. The plates were shaken thoroughly to completely dissolve the purple formazan crystals. The UV absorbance at 490 nm was recorded using a microplate reader, and the toxicity of the channel to HCT-116 cells was calculated. Cell viability was calculated based on the formula for adding different concentrations of the channel molecule.

[0085] To measure the cytotoxicity of the photoactivated channel, HCT-116 cells were incubated with the channel for 4 hours, followed by irradiation with a 24W LED lamp at 415 nm for 10 minutes. The HCT-116 cells were then incubated for another 20 hours at 37°C with 5% CO2, followed by the standard MTT assay described above. Some results obtained through software plotting are shown below. Figure 6 .

[0086] like Figure 6 As shown, C4F-L exhibits some cytotoxicity against HCT-116, but the activity of C4F-L modified with protecting groups is significantly reduced in the absence of visible light irradiation (IC50). 50 >100μM), while C4F-L showed an activity increase of more than 11 times after visible light activation (IC). 50=8.7 μM). To test the selectivity of the C4-L compound, the inhibition rate against normal HIEC-6 intestinal cells was tested in the absence of light. The results are as follows. Figure 7 As shown, this indicates that the channel molecules have almost no toxicity to normal cells (IC50). 50 >200μM).

[0087] Research on the anti-cancer mechanism of the channel

[0088] Flow cytometry

[0089] HCT-116 cells were seeded in 6-well plates at a density of 6 × 10⁶ cells per well. 5 Then, after incubating with different concentrations of C4F-L for 4 hours, the cells were irradiated with an LED lamp (24W) at a wavelength of 415 nm for 10 minutes. The HCT-116 cells were then incubated for another 20 hours. Cells were separated from the plates using 0.25% trypsin (1.0 mL) and resuspended in culture medium (1.0 mL) before centrifugation (112×g, 5 min). Cells were washed twice with PBS (2.0 mL) and stained in the dark for 15 minutes with annexin V-FITC (5 μL) and propidium iodide (PI, 5 μL). The percentage of apoptotic cells was determined by flow cytometry. Data were analyzed using software. Experimental results are as follows: Figure 8 As shown, the compound can induce apoptosis in HCT-116 cells in a concentration-dependent manner, with apoptosis reaching 20% ​​at 20 μM.

[0090] Changes in mitochondrial membrane potential and ROS content

[0091] HCT-116 cells were seeded in 6-well plates at a cell density of 1 × 10⁻⁶ cells per well. 6 The cells were then incubated with different concentrations of C4F-L (0, 5, 10 μM) for 4 hours, followed by irradiation with an LED lamp (24W) at 415 nm for 10 minutes. HCT-116 cells were then incubated for another 8 hours. Cells were washed twice with PBS (2.0 mL) and stained with 1.0 mL of 1×JC-1 staining working solution for 20 minutes. After washing twice with 1×JC-1 staining buffer, 1 mL of DMEM was added. Fluorescence images were acquired using a laser confocal microscope. The results are as follows: Figure 9 As shown, the compound can induce mitochondrial membrane potential depolarization in HCT-116 cells in a concentration-dependent manner.

[0092] HCT-116 cells were seeded in 6-well plates at a density of 6 × 10⁶ cells per well. 5HCT-116 cells were then incubated with different concentrations of C4F-L for 4 hours, followed by light irradiation using an LED lamp (24 W) at 415 nm wavelength for 10 minutes. HCT-116 cells were then incubated for another 20 hours. Subsequently, cells were washed once with PBS (2.0 mL) and lysed in lysis buffer containing 250 mM NaCl, 20 mM Tris-HCl (pH 7.4), ethylenediaminetetraacetic acid (EDTA, Solarbio), and Triton X-100 at 4 °C for 30 min. The concentration of protein was determined using a BCA kit. Next, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF, Millipore) membrane. After blocking with 5% skim milk, the blot was incubated in the primary antibody solution at 4 °C overnight. After washing with 1 x TBST for 3 times, the blot was incubated with the peroxidase-labeled secondary antibody solution (Abeam) at 25 °C for 2 hours, followed by washing with 1 x TBST solution. Finally, color development was performed using Pierce ECL Western Blotting Substrate and visualized imaging was performed using a chemiluminescence imager. Figure 10 As shown in FIG. 6, the compound was able to cause an increase in ROS content in HCT-116 cells in a concentration-dependent manner.

[0093] Western blot method for determining protein expression

[0094] HCT-116 cells were seeded in 6-well plates at a cell density of 6 x 10 5 HCT-116 cells were then incubated with different concentrations of C4F-L for 4 hours, followed by light irradiation using an LED lamp (24 W) at 415 nm wavelength for 10 minutes. HCT-116 cells were then incubated for another 20 hours. Subsequently, cells were washed once with PBS (2.0 mL) and lysed in lysis buffer containing 250 mM NaCl, 20 mM Tris-HCl (pH 7.4), ethylenediaminetetraacetic acid (EDTA, Solarbio), and Triton X-100 at 4 °C for 30 min. The concentration of protein was determined using a BCA kit. Next, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF, Millipore) membrane. After blocking with 5% skim milk, the blot was incubated in the primary antibody solution at 4 °C overnight. After washing with 1 x TBST for 3 times, the blot was incubated with the peroxidase-labeled secondary antibody solution (Abeam) at 25 °C for 2 hours, followed by washing with 1 x TBST solution. Finally, color development was performed using Pierce ECL Western Blotting Substrate and visualized imaging was performed using a chemiluminescence imager.

[0095] As shown in FIG. 6, the compound was able to cause an increase in ROS content in HCT-116 cells in a concentration-dependent manner. Figure 11 As shown in FIG. 6, the compound was able to cause an increase in ROS content in HCT-116 cells in a concentration-dependent manner.

[0096] Artificial ion channel affects autophagy by increasing the pH inside lysosomes

[0097] HCT-116 cells were seeded in 6-well plates at a cell density of 1 x 10 6 After incubation with different concentrations of C4F-L (0, 5, 10 μΜ) for 4 hours, the HCT-116 cells were then irradiated with an LED lamp (24 W) at a wavelength of 415 nm for 10 minutes. The HCT-116 cells were then incubated for an additional 8 hours. The cell pellets were washed twice with PBS (2.0 mL) and stained with 1.0 mL of AO staining solution (15 μg / mL) for 20 minutes. Finally, the cells were washed twice with PBS (2.0 mL) and 1.0 mL of DMEM was added. Fluorescence images were acquired using a laser confocal microscope.

[0098] The results, as shown in Figure 12 Figure 6, indicate that C4F-L is able to raise lysosomal pH and that immunoblotting shows upregulation of autophagy-related proteins LC3-II and p62, indicating that the artificial ion channel is able to interfere with the autophagy process.

Claims

1. A compound, characterized in that, Structural formula as follows:

2. Use of the compound of claim 1 in the preparation of an anticancer drug.

3. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable adjuvant.

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