Preparation method and application of active oxygen response type artificial ion channel

Abstract

According to the active oxygen response type artificial ion channel, high-level ROS in cancer cells is specifically activated, the activated channel promotes synergistic transmembrane transmission of H < + > and Cl <-> ions, so that specific anti-cancer treatment is achieved, the synthetic route is environmentally friendly, simple and efficient, raw materials are easy to obtain, and the active oxygen response type artificial ion channel is suitable for industrial production. The method can be used for synthesizing similar compounds.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the preparation of an active oxygen responsive artificial ion channel and its specific anti-cancer application. Background Art

[0002] Cancer is a major threat to human health. With the aging of my country's population, the situation will become more severe. At present, commonly used chemotherapy drugs in clinical practice, such as doxorubicin, paclitaxel, cisplatin, etc., generally have no cell specificity and produce obvious toxic side effects when used. In addition, the drug resistance problem of anticancer drugs also seriously affects the effect of chemotherapy. Therefore, the development of anticancer drugs based on new anticancer mechanisms and that can specifically act on tumor tissues has become a major issue in improving human health.

[0003] Due to abnormal metabolism, cancer cells have more intracellular reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radicals (·OH), superoxide (O2·-), singlet oxygen ( 1 For example, the H2O2 concentration in normal cells is around 20nM, while the H2O2 concentration in cancer cells is as high as 50-100μM, a difference of more than 2000 times.

[0004] Studies have found that artificial ion channels can disrupt the ion balance inside and outside the cancer cell membrane, thereby inducing cancer cell apoptosis, and are expected to be developed into a class of anticancer drugs based on a new mechanism of action. However, how to construct artificial ion channels so that they can act on tumor tissues with high specificity still faces huge challenges. Cancer cells are characterized by high expression of intracellular reactive oxygen species (ROS), which is caused by various factors such as mitochondrial dysfunction, oncogene activation, and abnormal metabolic processes. In addition, compared with normal cells, there are significant H + and Cl - At the same time, by taking advantage of the high level of ROS and H in the inherent oxidative microenvironment of cancer cells, + / Cl - The potential of ion co-transporters to further increase ROS and develop ROS-responsive artificial H + / Cl - Ion channels are expected to provide a new strategy for targeted cancer therapy.

[0005] The present invention is based on the structural characteristics of benzimidazole having both lone pair electrons and polar NH bonds, and utilizes its lone pair electrons and polar hydrogen as H + and Cl - Then, artificial H was constructed by one-dimensional self-assembly of imidazole units through intermolecular hydrogen bonds. + / Cl -channel, and further by introducing reactive oxygen species responsive groups, taking advantage of the huge difference in reactive oxygen content between cancer cells and normal cells, using reactive oxygen species responsive artificial H + / Cl - Achieve highly specific anti-cancer treatment.

[0006] SUMMARY OF THE INVENTION

[0007] In order to overcome the shortcomings of the prior art, the present invention provides a new artificial H + / Cl - The preparation of ion channels can be specifically activated by the high levels of ROS in cancer cells. The activated channels promote H + and Cl- ions through coordinated transmembrane transport, thereby achieving specific anti-cancer therapy.

[0008] The technical solution adopted by the present invention is mainly divided into four parts: one is artificial H + / Cl - Preparation of ion channels; second, study of the activity and selectivity of ion transport across the membrane of the channel; third, study of the ion transport mechanism of the channel; fourth, the production of artificial H2O2 responsive to reactive oxygen species + / Cl - Preparation of ion channels and study of their anticancer activity and mechanism.

[0009] The present invention has active oxygen responsive artificial H + / Cl - The general formula of the channel structure of ion channels used for specific anti-tumor is as follows:

[0010]

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

[0012]

[0013] The specific synthesis process is as follows: (1) 3-(3-nitrophenyl)propionic acid 1a and o-phenylenediamine 1b are used as raw materials, and 1c containing a phenylimidazole structure is condensed and cyclized in the presence of phosphorus oxychloride at high temperature. Then, the nitro group is reduced under the conditions of palladium carbon / hydrogen to obtain amine 1d, which is then reacted with different para-substituted isocyanates to form a urea structure, thereby preparing a new type of artificial H + / Cl - Ion channel CR (R = F, Cl, Br, CF3, CH3, C2H5, C4H9, OCH3, OC2H5). (2) Modification of the channel molecule in response to active oxygen. First, 4-(Hydroxymethyl)phenylboronic acid pinacol ester was reacted with p-nitrophenyl chloroformate to prepare intermediate 2c. Then, the artificial H + / Cl -Ion channel CR reacted with intermediate 2c in the presence of NaH to prepare a class of reactive oxygen species-responsive artificial H + / Cl - Ion channel ROS-CR (R=F,Cl,Br,CF3).

[0014] The compound obtained by the above preparation method is used in specific anti-cancer treatment.

[0015] A pharmaceutical composition comprises the compound obtained by the above preparation method and pharmaceutical excipients.

[0016] The beneficial effects of this application are:

[0017] 1. The synthetic route of the present invention is green, environmentally friendly, simple and efficient, and the raw materials are readily available, and can be used for the synthesis of similar compounds.

[0018] 2. The compound prepared by the present invention can induce apoptosis of human breast cancer cells (MCF-7) and has selectivity for liver cancer cells compared to normal breast cells (MCF-10A), with a selectivity index (SI) of 10.2, which is 20.4 times that of paclitaxel. The reactive oxygen responsive channel molecule prepared by the present invention can exert a specific anti-cancer effect in cancer cells.

[0019] 3. The reactive oxygen responsive channel molecules prepared by the present invention can cause depolarization of mitochondrial membrane potential and increase the ROS content and decrease the GSH content in cancer cells, and trigger the release of cytochrome c from mitochondria to the cytoplasm. The compounds prepared by the present invention can lead to the downregulation of typical anti-apoptotic proteins MCL1 and BCL2 and affect the expression levels of proteins such as Cleaved Caspase 9 and Cleaved PARP, triggering the Caspase 9 apoptosis pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the transmembrane transport activity of the channel molecule CR (2 μM);

[0021] Figure 2 EC for the transmembrane transport of C-Cl ions 50 Value determination;

[0022] Figure 3 Schematic diagram of the study on the selectivity of C-Cl ion transport across membranes;

[0023] Figure 4 Schematic diagram of the transmembrane transport rate of C-Cl ions;

[0024] Figure 5 Schematic diagram of the chloride ion transmembrane transport activity of C-Cl;

[0025] Figure 6In the figure, a) and c) are single channel current traces of C-Cl; b) and d) are recorded current-voltage (IV) diagrams;

[0026] Figure 7 Figure 3 shows the C-Cl diagram of the active channel molecules released by reactive oxygen species stimulating ROS-C-Cl;

[0027] Figure 8 This is a picture of ROS-C-Cl-induced apoptosis of MCF-7 cells;

[0028] Fig. 9 ROS-C-Cl caused the depolarization of mitochondrial membrane potential and the increase of ROS content in MCF-7 cells;

[0029] Fig.10 Schematic diagram of the release of cytochrome c caused by ROS-C-Cl;

[0030] Fig.11 ROS-C-Cl induced the expression of apoptosis-related proteins in MCF-7 cells;

[0031] Fig.12 ROS-C-Cl induces autophagy in MCF-7 cells. DETAILED DESCRIPTION

[0032] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0033] Embodiment one:

[0034] The preparation of an artificial H+ / CI- ion channel responsive to visible light and having specific anti-tumor activity is as follows:

[0035] Compound synthesis method:

[0036]

[0037] The precipitate was washed with water and then suspended in 5% NaOH for 30 minutes to remove any unreacted acid. The final product 1c was isolated by filtration, washed with water, and recrystallized from ethanol to obtain the pure product. Yield: 575 mg, 83%. 1H NMR (600MHz, DMSO-d6): δ12.61(s,1H),8.17(d,J=2.4Hz,1H),8.05(d,J=8.2Hz,1H),7.73(d,J=7.6Hz,1H),7.57(t,J= 7.9Hz, 1H), 7.48 (dd, J=6.3, 3.2Hz, 2H), 7.12 (dq, J=6.3, 3.9, 2.8Hz, 2H), 3.29 (t, J=7.7Hz, 2H), 3.20 (t, J=7.7Hz, 2H). 13 C NMR(151MHz,DMSO-d6)δ154.3,148.3,143.8,135.8,130.2,123.5,121.7,121.6,33.0,30.3.MS-ESI:calculatedfor[M+H] + (C 15 H 14 N3O2): m / z 268.1084, found: m / z 268.1083.

[0038]

[0039] 1d (300 mg, 1.3 mmol) was mixed with an equimolar amount of 1-chloro-4-isocyanatobenzene (200 mg, 1.3 mmol), and the resulting mixture was dissolved in a mixed solvent consisting of 10 mL of tetrahydrofuran, 5 mL of ethyl acetate and 5 mL of acetonitrile, and reacted at 80°C for 2 hours. The reaction solution was evaporated by conventional rotary evaporation under vacuum to obtain a crude white powder product. The crude product was washed with acetonitrile and hexane, and then filtered to separate the product C-Cl. Yield: 440 mg, 87%. 1 H NMR (600MHz, DMSO-d6) δ12.25(s,1H),8.79(d,J=2.1Hz,1H),8.65(d,J=2.1Hz ,1H),7.52(s,1H),7.48(dd,J=8.8,2.2Hz,2H),7.42(s,1H),7.40(q,J=2.0Hz ,1H),7.32(dt,J=8.8,2.7Hz,2H),7.29–7.25(m,1H),7.18(td,J=7.8,2.1Hz, 1H),7.11(d,J=6.9Hz,2H),6.88(d,J=7.5Hz,1H),3.10(tt,J=6.9,3.1Hz,4H). 13C NMR (151MHz, DMSO-d6) δ154.7,152.8,143.7,142.1,140.0,139.2,129.2,129.0,125.7,122.4,120.1,118.6,116.6,40.5,33.9,30.8.MS-ESI: calculated for[M+H] + (C 22 H 19 ClN4O): m / z 391.1320, found: m / z 391.1322.

[0040] 1d was reacted with different para-substituted isocyanates to generate the remaining CR compounds with urea structure and Control2.

[0041]

[0042] (td,J=7.8,2.5Hz,1H),7.12(dq,J=5.9,3.4,2.9Hz,2H),6.89(d,J=7.5Hz,1H),3.10(p,J=3.7Hz,4H). 13 C NMR(151MHz,DMSO-d6)δ154.7,152.8,142.1,140.0,139.6,131.9,129.2,122.4,120.5,118.6,116.6,113.6,33.9,30.8.MS-ESI:calculated for[M+H] + (C 22 H 20 BrN4O): m / z 435.0815, found: m / z 435.0819.

[0043]

[0044] 3.11(dq,J=8.5,5.7,4.7Hz,4H). 13 C NMR (151MHz, DMSO-d6) δ154.8,152.7,144.0,142.2,139.8,129.3,126.5,122.7,122.3,122.1,121.6,118.8,118.3,116.8,34.0,30.9.MS-ESI: calculated for[M+H] + (C 23 H 20F3N4O):m / z 425.1584,found:m / z425.1585.MS-ESI:calculated for[M+H] + (C 23 H 20 F3N4O):m / z 425.1584,found:m / z425.1585。

[0045]

[0046] 13 C NMR(151MHz,DMSO-d6)δ158.5,154.7,153.0,142.1,140.1,136.5,129.2,122.2,121.6,120.4,120.3,118.5,116.5,115.8,115.6,33.9,30.8.MS-ESI:calculatedfor[M+H] + (C 22 H 20 FN4O):m / z 375.1616,found:m / z 375.1616。

[0047]

[0048] 7.8 Hz,1H),7.12(q,J=4.3 Hz,2H),7.08(d,J=8.1 Hz,2H),6.86(d,J=7.5Hz,1H),3.10(tt,J=6.9,4.1 Hz,4H),2.24(s,3H). 13 C NMR(151 MHz,DMSO-d6)δ154.8,153.0,143.7,142.0,140.3,137.6,134.7,131.0,129.6,129.2,122.1,121.9,121.2,118.7,118.6,118.4,116.4,111.2,34.0,30.8,20.8.MS-ESI:calculated for[M+H] + (C 23 H 22 N4O):m / z 371.1866,found:m / z371.1889。

[0049]

[0050] J=8.5,6.0 Hz,4H),6.86(dt,J=7.6,1.4 Hz,1H),3.12–3.05(m,4H),2.54(q,J=7.6 Hz,2H),1.16(t,J=7.6 Hz,3H). 13 C NMR(151 MHz,DMSO-d6)δ154.8,153.0,143.7,142.0,140.3,137.8,137.6,134.7,129.2,128.4,122.1,121.9,121.2,118.7,118.6,118.4,116.4,111.2,34.0,30.8,27.9,16.2.MS-ESI:calculated for[M+H] + (C 24 H 25 N4O):m / z 385.2023,found:m / z385.2046。

[0051]

[0052] 7.08(d,J=2.0 Hz,1H),6.86(dt,J=7.6,1.3 Hz,1H),3.14–3.05(m,4H),2.52–2.49(m,2H),1.55–1.49(m,2H),1.29(h,J=7.4 Hz,2H),0.89(t,J=7.4 Hz,3H). 13 C NMR(151 MHz,DMSO-d6)δ154.8,153.0,142.1,140.3,137.7,136.1,129.2,128.9,122.1,118.7,118.4,116.4,34.6,34.0,33.7,30.8,22.1,14.2.MS-ESI:calculated for[M+H] + (C 26 H 29 N4O):m / z 413.2336,found:m / z 413.2360。

[0053]

[0054] (dq,J=6.1,4.1,3.0 Hz,3H),3.72(d,J=1.9 Hz,3H),3.09(h,J=7.5 Hz,4H). 13C NMR(151MHz,DMSO-d6)δ154.9,154.8,153.1,142.0,140.4,133.2,129.2,122.0,120.4,118.3,116.4,114.4,55.6,34.0,30.8.MS-ESI:calculated for[M+H] + (C 23 H 23 N4O2):m / z 387.1816,found:m / z387.1839。

[0055]

[0056] 7.2,4.1 Hz,2H),6.88–6.81(m,3H),3.97(q,J=6.9 Hz,2H),3.14–3.03(m,4H),1.31(t,J=6.9 Hz,3H). 13 C NMR(151 MHz,DMSO-d6)δ154.8,154.1,153.1,142.0,140.4,133.1,129.2,122.0,120.4,118.3,116.4,115.0,63.5,34.0,30.8,15.2.MS-ESI:calculated for[M+H] + (C 24 H 25 N4O2):m / z 401.1972,found:m / z 401.1999。

[0057]

[0058] calculated for[M+H] + (C 11 H 16 ClN2O):m / z 227.0946,found:m / z 227.0949.

[0059]

[0060] 7.44(dd,J=8.1,2.3 Hz,2H),7.40–7.35(m,2H),5.31(d,J=2.3 Hz,2H),1.37–1.34(m,12H). 13C NMR(151 MHz,Chloroform-d)δ155.6,152.5,145.5,137.1,135.2,127.7,125.3,121.8,84.0,70.9,24.9.MS-ESI:calculated for[M+H] + (C 20 H 23 BNO7):m / z400.1562,found:m / z 400.1843。

[0061]

[0062] Hz,1H),7.75–7.71(m,2H),7.67(dt,J=5.8,2.1 Hz,1H),7.60–7.56(m,2H),7.51–7.45(m,2H),7.37(d,J=2.2 Hz,1H),7.33(dtd,J=9.0,7.3,6.6,4.8 Hz,5H),7.17(td,J=7.8,1.8 Hz,1H),6.78(d,J=7.5 Hz,1H),5.56(d,J=1.9 Hz,2H),3.41(ddd,J=10.3,6.2,1.8 Hz,2H),3.07(t,J=8.1 Hz,2H),1.30(d,J=1.9 Hz,12H). 13 C NMR(151MHz,DMSO-d6)δ155.95,152.87,150.28,142.11,140.04,139.22,138.39,135.22,129.22,129.06,128.47,125.74,124.84,124.68,122.43,120.14,119.72,118.63,116.58,115.25,84.23,69.53,33.19,32.72,25.13.MS-ESI:calculated for[M+Na] + (C 36 H 36 BClN4O5Na):m / z651.2540,found:m / z 651.2546。

[0063]

[0064] 140.0,138.4,135.2,132.0,129.2,128.5,124.8,124.5,122.5,120.6,119.7, 118.6,116.6,115.2,113.6,84.2,69.5,33.2,32.7,25.1.MS-ESI:calculated for[M+Na] + (C 36 H 36 BBrN4O5Na): m / z717.1854, found: m / z 717.1859.

[0065]

[0066] 116.7,115.2,84.2,69.5,33.2,32.7,25.1.MS-ESI:calculated for[M+Na] + (C 37 H 36 BF3N4O5Na): m / z 685.2804, found: m / z 685.2805.

[0067]

[0068] MS-ESI: calculated for[M+H] + (C 36 H 37 BFN4O5): m / z 635.2836, found: m / z 635.2837.

[0069] Experimental Example 1

[0070] Transmembrane transport activity assay of compounds

[0071] Ion transport based on HPTS vesicle fluorescence experiments

[0072] 1 mL of egg yolk phosphatidylcholine (EYPC, 25 mg / mL) dissolved in chloroform solution was placed in a round-bottom flask, chloroform was removed under reduced pressure at 35°C, and dried overnight under vacuum. 1 mL of pH-sensitive HTPS (0.1 mM, pH = 7.0, 100 mM MCl, HEPES 10 mM) buffer solution was added to the round-bottom flask and shaken for hydration at 37°C for 2 hours. The resulting solution was placed in a water bath at room temperature after being frozen in liquid nitrogen for 1 minute and heated for 2 minutes. After 10 freeze-thaw cycles, the suspension was extruded 21 times through a polycarbonate membrane with a pore size of 100 nm to obtain a uniform size vesicle (LUV) suspension. Finally, the uncoated fluorescent dye in the LUV suspension was dialyzed using a dialysis membrane to produce LUVs with a lipid concentration of 6.5 mM.

[0073] 30 μL of LUV suspension coated with HPTS was added to 1.95 mL of NaCl buffer solution (pH = 8.0, 100 mM NaCl, HEPES 10 mM). After adding different concentrations of channel compound molecules under stirring, the fluorescence intensity change of the solution at 510 nm emission wavelength was detected by fluorescence spectrophotometer under dual wavelength excitation of 403 nm and 460 nm for 300 seconds. The final transmission curve was calculated by I 460 / I 403 The ratio of I was calculated by equation (1) after adding Triton X-100. 460 / I 403 The ratio values ​​are normalized.

[0074] I f =[(I t -I0) / (I1-I0)] (1)

[0075] 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.

[0076] The EC was obtained by fitting the curve of ion channel concentration to ion transport activity R using Hill (S2). 50 Value and Hill coefficient n.

[0077] R=1 / (1+(EC 50 / [aisle]) n ) (2)

[0078] like Figure 1As shown in the figure, the results of the HPTS experiment show that the ion transport activity of CR is affected by different substituents. C-Cl showed the highest activity. After background correction, C-Cl showed 93% ion transport activity at a concentration of 2 μM. By measuring the transport activity of C-Cl at different concentrations, its corresponding EC 50 The value is 0.21μM ( Figure 2 ).

[0079] Figure 3 The description is that by changing the alkali metal ion species (M + =Li + ,Na + ,K + ,Rb + Cs + ), the ion transport activity of C-Cl remained basically unchanged, indicating that alkali metal ions did not participate in the ion transport process. Combined with the molecular structure characteristics, it can be ruled out that Na + / OH - Cotransport and Na + / H + Antiport is the main mechanism of ion transport. Therefore, the main mechanism of C-Cl-facilitated ion transport process is Cl - / H + Cotransport or Cl - / OH - Reverse transport.

[0080] In the HPTS assay, we further used gramicidin (GA), a highly active proton transporter, to compare Cl - and H + The ion transfer rate between + Selective transporter - valinomycin (VA) compared with Cl - and OH - The transport rate of ions.

[0081] like Figure 4 As shown in a, C-Cl (0.21 μM) exhibited almost the same ion transport activity in the presence and absence of GA (100 pM), which indicates that H + or OH - The transmission speed is faster than Cl - quick. Figure 4 b shows that VA and C-Cl (0.21 μM) did not show synergistic effect, indicating that Cl - Transmission speed is faster than OH - Fast. Combining the results of the two experiments, we can conclude that: + / Cl - Symport is the main transport mechanism, and H +The transport rate is faster than Cl - Come on, Cl - The transport rate of OH - quick.

[0082] Ion transport based on SPQ vesicle fluorescence experiments

[0083] 1 mL of egg yolk phosphatidylcholine (EYPC, 25 mg / mL) dissolved in chloroform solution was placed in a round-bottom flask, chloroform was removed under reduced pressure at 35°C, and dried overnight under vacuum. 1 mL of NaNO3 solution (200 mM) containing 6-methoxy-N-(3-sulfopropyl)quinoline (SPQ) (0.5 mM), a dye sensitive to chloride ions, was added to the round-bottom flask and shaken for hydration at 37°C for 2 hours. The resulting solution was placed in a water bath at room temperature after being frozen in liquid nitrogen for 1 minute and heated for 2 minutes. After 10 freeze-thaw cycles, the suspension was extruded 21 times through a polycarbonate membrane with a pore size of 100 nm to obtain a uniform vesicle suspension. Finally, the uncoated fluorescent dye in the LUV suspension was dialyzed using a dialysis membrane to produce LUVs with a lipid concentration of 6.5 mM.

[0084] 30 μL of LUV suspension coated with SPQ was added to 1.95 mL of NaCl (200 mM) buffer solution to generate a chloride ion gradient inside and outside the vesicles. After adding different concentrations of channel molecules under stirring, the fluorescence intensity change of the solution at an emission wavelength of 430 nm was detected by a fluorescence spectrophotometer at an excitation wavelength of 360 nm for 300 seconds. The final transmission curve was calculated by I 460 / I 403 The ratio value was calculated and normalized by the above equation (1) after adding Triton X-100. The curve of ion channel concentration versus ion transport activity R was fitted using the above equation (2) to obtain EC 50 Value and Hill coefficient n.

[0085] like Figure 5 As shown in the figure, when C-Cl was added to the LUV containing the chloride-sensitive dye SPQ, the fluorescence intensity of SPQ was observed to show a concentration-dependent quenching, and the EC 50 The value as low as 0.07 μM indicates that C-Cl promotes the Cl - Ion transport across the membrane and the trend of chloride transport activity remained consistent with that in the HPTS assay.

[0086] Experiments on planar lipid bilayers demonstrate that compounds are transported via an adaptive channel mechanism

[0087] Measuring chloride ion conductivity (γ): Take 20 μL of DiPhyPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine, 10 mg / mL) phospholipid chloroform solution in a sample bottle and blow it dry with nitrogen gas, then redissolve it in 8 μL of n-decane. Inject 0.5 μL of n-decane solution containing lipid into Cup (Warner Instruments, Hamden, CT) hole (diameter = 200μm), N2 blow dry to remove n-decane. In the patch clamp experiment, KCl solution (1.0M, 1.0mL) was added to the chamber (cis side) and the Delrin cup (transverse side). Ag-AgCl electrodes were inserted into the two solutions, and the cis chamber was grounded. A planar lipid bilayer was formed by applying 0.5μL of lipid-containing n-decane solution around the n-decane pretreated hole. When the capacitance value is in the range of 80-120pF, it indicates that a planar lipid bilayer has been formed. Samples in DMSO (1.0μL) were added to the cis compartment to reach a final concentration of about 1μM. These single-channel currents were then measured using a Warner BC-535D double-layer clamp amplifier, collected by PatchMaster (HEKA) at a sampling interval of 5kHz and filtered using an 8-pole Bessel filter at 1kHz (HEKA). The data were analyzed by FitMaster (HEKA) and a 100Hz digital filter. Plotting the current trajectory versus voltage yields the chloride conductance (γ).

[0088] Measuring H + / Cl - Ion transfer selectivity: Monoolein (15.0 mg) and cholesterol (15 mg) were dissolved in 0.5 ml chloroform / methanol (2 / 1, v / v) to prepare lipid stock solution. 50 μL of lipid-containing solution was added to the sample vial along with the channel solution in methanol. The solution was evaporated in N2 to form a thin film, which was redissolved in 50 μL n-decane to produce a channel to GMO molar ratio of 1 / 10. 6 Inject 0.5 μL of lipid-containing n-decane solution into The pore size of the cup (Warner Instruments, Hamden, CT) was 200 μm, and the n-decane was removed using N2. In the experiment, the cis chamber was filled with 0.05 M HCl and the trans chamber was filled with 0.025 M HCl. The single-channel current was recorded at different voltages. The channel was responsive to H + Relative to Cl - The selectivity is defined as the ratio of the permeability of the two ions (P H+ / P Cl- ), can be obtained by fitting the IV curve using the following simplified Goldman-Hodgkin-Katz equation (3).

[0089] ε rev +17.8=RT / F×ln{(P H+ [H + ] trans +P Cl- [Cl - ] cis ) / (P H+ [H + ] cis +P Cl- [Cl - ] trans )} (3)

[0091] where ε rev is the reversal membrane potential; 17.8 mV is the Nernst potential corresponding to a proton gradient of 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); and P is the permeability of the ion channel.

[0092] like Figure 6 As shown, C-Cl mediates H transport via a channel mechanism rather than a carrier mechanism. + / Cl - The Cl of C-Cl was determined by linear fitting of the current-voltage (IV) graph. - Conductivity (γ Cl- ) is 15.6±0.1ps. In addition, the H + / Cl - Transmission selectivity (P H+ / P Cl- ) was calculated to be 7.7, further confirming that H + The transmission speed is faster than Cl - quick.

[0093] Experimental Example 2

[0094] Reactive oxygen species stimulate the release of active channel molecules

[0095] like Figure 7 As shown in a, the introduction of the active oxygen-responsive phenylboronic acid group significantly reduced the ion transport activity. ROS-C-Cl can restore 90% of the transmembrane transport activity at low concentrations with only 5 equivalents of H2O2 stimulation. High-performance liquid chromatography was used to evaluate the ability of ROS-C-Cl to release the active channel molecule C-Cl in the presence of H2O2, as shown in Figure 7As shown in b, the HPLC spectrum of ROS-C-Cl (100 μM) changed significantly after incubation with different concentrations of H2O2 for 30 minutes. A new retention peak corresponding to the parent channel forming unit C-Cl appeared, and the concentration-dependent decrease of the ROS-C-Cl signal was observed. After 30 minutes of treatment with 5 equivalents of H2O2, the conversion ratio of ROS-C-Cl to C-Cl was 93%.

[0096] Experimental Example 3

[0097] Anticancer activity and anticancer mechanism detection of compounds

[0098] Anticancer activity and selectivity studies

[0099] In a 96-well plate, each well was inoculated with 10 4 Breast cancer cells (MCF-7) and normal breast cells (MCF-10A) were cultured overnight in an incubator containing 5% CO2 at 37°C. The samples were dissolved in DMSO to prepare a series of solutions of different concentrations, and then selected and added to 96-well plates containing cells and incubated at 37°C for 48 hours. 20 μL of MTT solution was added to the culture medium, and the fluorescence of the solution was detected with an enzyme marker to measure cell activity, and the anticancer activity of the channel molecule on MCF-7 and its safety were calculated. The anticancer activity IC of ROS-CR on MCF-7 cells and MCF-10A cells 50 The values ​​and selectivity index are summarized in Table 1.

[0100] Table 1. IC of ROS-CR against human MCF-7 and MCF-10A cells 50 Value and selectivity index

[0101]

[0102] All four ROS-CRs showed good anticancer activity. The most active molecule, ROS-CR, had anti-breast cancer activity similar to paclitaxel (IC 50 =2.8 μM). ROS-C-Cl showed excellent specificity for breast cancer cells and a selectivity index of 10.2 for normal liver cells. Compared with paclitaxel, ROS-C-Cl was 20.2 times more specific for cancer cells.

[0103] Flow cytometry to detect cell apoptosis

[0104] Inoculate ~10 cells per well in a 6-well plate. 5MCF-7 cells. After culturing in an incubator at 37°C and 5% CO2 for 12 hours, different concentrations of ROS-C-Cl (0, 5, 10 μM) were added to treat the cells for 36 hours. After the culture medium was removed and the cells were washed with PBS, they were digested with EDTA-free trypsin and the cell suspension was collected. 5 μL of FITC-Annexin-V and 10 μL of PI solution were added, and the cells were incubated at room temperature in the dark for 15 minutes. The apoptosis of tumor cells was detected by flow cytometry. Figure 8 As shown, as the concentration of ROS-C-Cl increased from 0 to 10 μM, the percentage of apoptotic cancer cells increased from 2.4% to 15.7%, confirming the ability of ROS-C-Cl to induce apoptosis in MCF-7 cells.

[0105] Changes in mitochondrial membrane potential and ROS content

[0106] Inoculate ~10 cells per well in a 6-well plate. 5 MCF-7 cells. After culturing in an incubator at 37°C and 5% CO2 for 12 hours, different concentrations of ROS-C-Cl (0, 5, 10 μM) were added to treat the cells for 24 hours. After aspirating the culture medium and washing the cells with PBS, they were 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. In normal mitochondrial membranes, JC-1 fluorescent dye exists in the form of J-aggregates, which mainly emit red fluorescence. When the mitochondrial membrane is depolarized, the formed aggregates will dissociate, thereby emitting green fluorescence. The experimental results are as follows. Fig. 9 As shown in a, ROS-C-Cl can increase the green fluorescence and decrease the red fluorescence of MCF-7 cells in a concentration-dependent manner, indicating that ROS-C-Cl can cause mitochondrial membrane potential depolarization.

[0107] Inoculate ~10 cells per well in a 6-well plate. 5 MCF-7 cells. After culturing in an incubator at 37°C and 5% CO2 for 12 hours, different concentrations of ROS-C-Cl (0, 5, 10 μM) were added to treat the cells for 6 hours. After aspirating the culture medium and washing the cells with PBS, the cell suspension was digested and collected with EDTA-free trypsin, and stained with 1.0 mL of reactive oxygen probe DCFH-DA staining working solution for 20 minutes, and the relative ROS level in the cells was detected by flow cytometry. The experimental results are shown in Fig. 9 As shown in b, ROS-C-Cl can increase the ROS content in MCF-7 cells in a concentration-dependent manner.

[0108] Cytochrome c release studies

[0109] MCF-7 cells were collected at 1×10 6The cells were transplanted onto glass coverslips at a density of cells / well and cultured overnight in a cell culture incubator. Then, different concentrations of ROS-C-Cl (0, 5, 10 μM) were added to treat the cells for 12 hours. The culture medium was aspirated and the cells were washed with PBS. The cells were then fixed with 4% formalin and permeabilized with 0.5% Triton-X at 4°C for 10 minutes. After blocking the cells with 10% (v / v) FBS, they were stained with cytochrome c antibody at 4°C overnight. After staining, the cells were washed with PBS and incubated with fluorescent secondary antibodies. Finally, the cell borders were stained with phalloidin, transferred to a glass slide, and the localization and fluorescence intensity of cytochrome c were observed using a laser confocal microscope. Fig.10 As shown, since the increase in ROS levels can cause changes in mitochondrial permeability, a significant increase in green fluorescence was observed after cells were treated with ROS-C-Cl, confirming the release of cytochrome c.

[0110] Apoptosis-related protein expression detection

[0111] MCF-7 cells were cultured at a cell density of 1×10 6 Inoculate in 6-well plates and culture in an incubator for 24h. After incubation for 48 hours with ROS-C-Cl (0, 5, 10, 20 μM), MCF-7 cells were lysed with RIPA buffer containing protease and phosphatase inhibitors (PMSF) for 20 minutes, and cell proteins were collected by centrifugation at 12,000 rpm for 10 minutes at 4°C. The protein concentration was then determined using a BCA assay kit. Take an equal amount of protein (20 μg) and heat it at 95°C for 5 minutes in SDS sample buffer for denaturation, and then separate it with a 10% SDS-page gel. After separation, the protein was transferred to a PVDF membrane. The membrane was blocked in TBST (TRIS buffered saline, containing 0.1% Tween-20) containing 5% skim milk at room temperature for 1 hour to prevent nonspecific binding. Then incubate with antibodies to apoptosis- and autophagy-related proteins at 4°C overnight. The cells were washed three times with TBST, each time for 10 min, and then incubated with horseradish peroxidase-labeled secondary antibody solution (Abcam) at room temperature at 25°C for 2 h, followed by washing with 1× TBST solution. Finally, the cells were developed with ECL reagent, observed with an exposure instrument, and photographed.

[0112] like Fig.11As shown, the above-mentioned immunoblotting method for apoptosis-related proteins showed that treatment of MCF-7 cells with ROS-C-Cl for 48 hours caused a concentration-dependent downregulation of anti-apoptotic proteins MCL1 and BCL2, indicating that ROS-C-Cl can promote apoptosis of MCF-7 cells through the mitochondrial pathway. The observed upregulation of pro-apoptotic proteins cleaved caspase9 and cleavedPARP further supported ROS-C-Cl-induced apoptosis, indicating that the apoptotic cascade was thoroughly activated.

[0113] Artificial ion channels increase lysosomal internal pH and induce autophagy

[0114] Inoculate ~10 cells per well in a 6-well plate. 5 MCF-7 cells. After culturing in an incubator at 37°C and 5% CO2 for 12 hours, different concentrations of ROS-C-Cl (0, 5, 10 μM) were added to treat the cells for 12 hours. After aspirating the culture medium and washing the cells with PBS, the cell suspension was collected by digesting with EDTA-free trypsin and stained with 1.0 mL of AO staining solution (15 μg / mL) for 20 minutes. After washing twice with PBS, 1 mL of DMEM was added. Fluorescence images were obtained using a laser confocal microscope. The results are shown in Fig.12 As shown, ROS-C-Cl could increase lysosomal pH and immunoblotting showed upregulation of autophagy-related proteins LC3-II and p62, indicating that ROS-C-Cl could interfere with the autophagy process.

[0115] Different from the existing tubular cavity and non-covalently stapled artificial H + / Cl - The present invention is based on a completely different molecular structure, using the structural characteristics of imidazole that has both lone pair electrons and polar NH bonds, and using lone pair electrons and polar hydrogen as H + and Cl - Then, the ion recognition units are assembled one-dimensionally through intermolecular hydrogen bonds to construct a system that can cooperatively transport H + and Cl - The artificial channel, H + / Cl - The ion transport selectivity is 7.7. In addition, unlike artificial H that responds to external stimuli such as visible light, + / Cl - channel, the present invention is to artificial H + / Cl - The channel introduces groups that respond to endogenous stimuli such as reactive oxygen species, so that it can only be activated under the stimulation of reactive oxygen species that are highly expressed in cancer cells. After activation, ROS-C-Cl activates the Caspase 9 apoptosis pathway and disrupts the autophagy process, IC 50Most importantly, by taking advantage of the inherent oxidative microenvironment of cancer cells and the + / Cl - Due to the enhanced oxidative stress caused by co-transport, ROS-C-Cl showed excellent selectivity in targeting breast cancer cells, with a selectivity index of 10.2 compared with normal breast cells. This selectivity greatly exceeded the selectivity of the traditional chemotherapy drug paclitaxel by 20.2 times, highlighting the potential of ROS-responsive artificial H + / Cl - Ion channels have great potential as cancer-specific therapeutic drugs.

Claims

1. An artificial ion channel responsive to active oxygen species, characterized in that: The general structure is as follows:

2. A method for preparing an active oxygen responsive artificial ion channel, characterized in that: The method comprises the following synthesis steps: (1) 3-(3-nitrophenyl)propionic acid 1a and o-phenylenediamine 1b were used as raw materials, and condensed and cyclized at high temperature in the presence of phosphorus oxychloride to generate 1c containing a phenylimidazole structure. The nitro group was then reduced under palladium carbon / hydrogen conditions to obtain amine 1d, which was then reacted with different para-substituted isocyanates to form urea structures, thereby preparing a new type of artificial H + / Cl - Ion channel CR (R = F, Cl, Br, CF3, CH3, C2H5, C4H9, OCH3, OC2H5); (2) Modification of channel molecules in response to active oxygen: First, 4-(Hydroxymethyl)phenylboronic acid pinacol ester was reacted with p-nitrophenyl chloroformate to prepare intermediate 2c, and then the artificial H + / Cl - Ion channel CR reacted with intermediate 2c in the presence of NaH to prepare a class of reactive oxygen species-responsive artificial H + / Cl - Ion channel ROS-CR (R=F,Cl,Br,CF3).

3. The method for preparing an active oxygen responsive artificial ion channel according to claim 2, characterized in that: The synthetic route is as follows:

4. Use of the compound obtained by the preparation method as claimed in claim 3 in specific anti-cancer treatment.

5. A pharmaceutical composition comprising the compound obtained by the preparation method of claim 3 and pharmaceutical excipients.

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