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

Abstract

The application provides an active oxygen response type artificial ion channel, and a structural general formula is as follows: the application is activated by high level ROS in cancer cells, the activated channel promotes the synergistic transmembrane transmission of H + and Cl ‑ ions, thereby realizing specific anticancer treatment, the synthetic route of the application is green, simple, efficient, raw materials are easy to obtain, and can be used for synthesis of similar compounds.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to preparation of an active oxygen response type artificial ion channel and specific anti-cancer application thereof. BACKGROUND

[0002] Cancer is a big threat to human health. With the aggravation of population aging in China, the situation will be more severe. At present, the commonly used clinical chemotherapy drugs such as doxorubicin, paclitaxel and cisplatin generally have no cell specificity, and obvious toxic side effects will be produced when used. In addition, the drug resistance of anti-cancer drugs also seriously affects the effect of chemotherapy. Therefore, developing anti-cancer drugs based on new anti-cancer mechanisms and capable of specifically acting on tumor tissues has become a major issue for improving human health.

[0003] Due to abnormal metabolism, the intracellular active oxygen (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (·OH), superoxide (O2·-) and singlet oxygen ( 1 O2) in cancer cells is obviously higher than that in normal cells. For example, the H2O2 concentration in normal cells is about 20 nM, while the H2O2 concentration in cancer cells is as high as 50-100 μM, which is more than 2000 times different.

[0004] It is found that artificial ion channels can induce cancer cell apoptosis by destroying the ion balance inside and outside the cancer cell membrane, and are expected to be developed into a new type of anti-cancer drugs based on new mechanisms. However, how to construct artificial ion channels to specifically act on tumor tissues still faces great challenges. The characteristics of cancer cells are high expression of intracellular active oxygen (ROS), which is caused by mitochondrial dysfunction, activation of oncogenes and abnormal metabolic processes and other factors. In addition, compared with normal cells, there is also a significant H + and Cl - gradient inside and outside the cancer cell membrane. At the same time, by taking advantage of the high level of ROS content and the potential of H + / Cl - ion co-transporter to further increase ROS in the inherent oxidative microenvironment of cancer cells, the development of ROS response type artificial H + / Cl - ion channels is expected to provide a new strategy for targeted cancer treatment.

[0005] Based on the structural characteristics of benzimidazole having lone pair electrons and polar N-H bond, the present application uses the lone pair electrons and the polar hydrogen as the recognition units of H + and Cl - respectively, and then constructs artificial H + / Cl -The channel, and further by introducing reactive oxygen species responsive groups, utilizes the huge difference in reactive oxygen species content between cancer cells and normal cells to utilize reactive oxygen species responsive artificial H channels. + / Cl - To achieve highly specific anti-cancer treatment.

[0006] Invention Content

[0007] To overcome the shortcomings of existing technologies, this invention provides a novel artificial H + / Cl - The ion channel preparation method utilizes the high levels of ROS in cancer cells for specific activation. The activated channel promotes H+ ionization. + It can achieve specific anticancer therapy through synergistic transmembrane transport with Cl- ions.

[0008] The technical solution adopted in this invention mainly consists of four parts, the first being artificial H + / Cl - The research focuses on four main areas: 1) preparation of ion channels; 2) study on the transmembrane ion transport activity and selectivity of these channels; 3) study on the ion transport mechanism of these channels; and 4) development of reactive oxygen species-responsive artificial H2O channels. + / Cl - Preparation of ion channels and research on their anticancer activity and mechanism.

[0009] The artificial H2O with reactive oxygen species responsiveness described in this invention + / Cl - The general structural formula of ion channels used for specific antitumor activity is as follows:

[0010]

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

[0012]

[0013] The specific synthesis process is as follows: (1) Using 3-(3-nitrophenyl)propionic acid 1a and o-phenylenediamine 1b as raw materials, a high-temperature reaction condensation and cyclization reaction is carried out in the presence of phosphorus oxychloride to generate 1c containing a benzylimidazole structure. Then, the nitro group is reduced under palladium carbon / hydrogen conditions to obtain amine 1d. Then, it is reacted with different para-substituted isocyanates to generate urea structures, thus preparing a new type of artificial H + / Cl - Ion channels CR (R=F,Cl,Br,CF3,CH3,C2H5,C4H9,OCH3,OC2H5). (2) Modification of channel molecules to respond to reactive oxygen species. First, intermediate 2c is prepared by reacting 4-(hydroxymethyl)phenylboronic acid pinacol ester with p-nitrophenyl chloroformate. Then, the artificial H prepared above is... + / Cl -The ion channel C-R is reacted with intermediate 2c in the presence of NaH to prepare a class of active oxygen response type artificial H + / Cl - Ion channel ROS-C-R (R=F, Cl, Br, CF3).

[0014] The use of the compound prepared by the above preparation method in specific anticancer treatment.

[0015] A pharmaceutical composition comprising the compound prepared by the above preparation method and a pharmaceutical adjuvant.

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

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

[0018] 2. The compound prepared by the present application can induce human breast cancer cell (MCF-7) apoptosis, and has selectivity to liver cancer cells compared with normal breast cells (MCF-10A), and the selectivity index (SI) is 10.2, which is 20.4 times that of paclitaxel. The active oxygen response type channel molecule prepared by the present application can play a specific anticancer effect in cancer cells.

[0019] 3. The active oxygen response type channel molecule prepared by the present application can cause depolarization of mitochondrial membrane potential and lead to an increase in ROS content and a decrease in GSH content in cancer cells, and release of cytochrome c from mitochondria to cytoplasm. The compound prepared by the present application can lead to down-regulation of typical anti-apoptotic proteins MCL1 and BCL2 and affect the expression levels of proteins such as Cleaved Caspase 9 and Cleaved PARP, and trigger the Caspase 9 apoptosis pathway. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a schematic diagram of the transmembrane transport activity of channel molecule C-R (2 mu M);

[0021] Figure 2 It is the EC value determination of the ion transmembrane transport activity of C-Cl. 50

[0022] Figure 3 It is a schematic diagram of the ion transmembrane transport selectivity study of C-Cl.

[0023] Figure 4 It is a schematic diagram of the ion transmembrane transport rate of C-Cl.

[0024] Figure 5 It is a schematic diagram of the ion transmembrane transport activity of C-Cl.

[0025] Figure 6 ​Fig. 1. a) and c) are single channel current traces of C-Cl; b) and d) are recorded current-voltage (I-V) plots;

[0026] Figure 7 Fig. 4. Scheme for ROS-C-Cl release of active channel molecule C-Cl;

[0027] Figure 8 Fig. 5. Scheme for ROS-C-Cl induction of MCF-7 cell apoptosis;

[0028] Figure 9 Fig. 6. Scheme for ROS-C-Cl induced depolarization of mitochondrial membrane potential and increase in ROS content in MCF-7 cells;

[0029] Figure 10 Fig. 7. Scheme for ROS-C-Cl induced release of cytochrome c;

[0030] Figure 11 Fig. 8. Scheme for ROS-C-Cl induction of MCF-7 cell apoptosis related protein expression;

[0031] Figure 12 Fig. 9. Scheme for ROS-C-Cl induction of MCF-7 cell autophagy. 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] A visible light-responsive artificial H+ / CI- ion channel with specific anti-tumor activity is prepared as follows:

[0035] Compound synthesis method:

[0036]

[0037] The precipitate was washed with water, then suspended in 5% NaOH for 30 minutes to remove any unreacted acid, and the final product 1c was separated by filtration, washed with water, and recrystallized in ethanol to obtain the pure product. Yield: 575 mg, 83%. 1H NMR (600 MHz, DMSO-d6) δ 12.61 (s, 1H), 8.17 (d, J = 2.4 Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.48 (dd, J = 6.3, 3.2 Hz, 2H), 7.12 (dq, J = 6.3, 3.9, 2.8 Hz, 2H), 3.29 (t, J = 7.7 Hz, 2H), 3.20 (t, J = 7.7 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 154.3, 148.3, 143.8, 135.8, 130.2, 123.5, 121.7, 121.6, 33.0, 30.3. MS-ESI: calculated for [M+H] + (C 15 H 14 N3O2): m / z 268.1084, found: m / z 268.1083.

[0038]

[0039] A mixture of 1d (300 mg, 1.3 mmol) and equimolar amount of 1-chloro-4- isocyanatobenzene (200 mg, 1.3 mmol) 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 removed from the solvent under vacuum using a conventional rotary evaporator to obtain a white powder crude product. The crude product was washed with acetonitrile and hexane, and then filtered to isolate the product C-Cl. Yield: 440 mg, 87%. 1 H NMR (600 MHz, DMSO-d6) δ 12.61 (s, 1H), 8.17 (d, J = 2.4 Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.48 (dd, J = 6.3, 3.2 Hz, 2H), 7.12 (dq, J = 6.3, 3.9, 2.8 Hz, 2H), 3.29 (t, J = 7.7 Hz, 2H), 3.20 (t, J = 7.7 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) d 154.7, 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] 391.1320, found: m / z 391.1322. + (C 22 H 19 ClN4O): m / z 391.1320, found: m / z 391.1322.

[0040] Reaction of 1d with different para-substituted isocyanates to generate the rest of C-R compounds with urea structure and Control 2.

[0041]

[0042] (td, J = 7.8, 2.5 Hz, 1 H), 7.12 (dq, J = 5.9, 3.4, 2.9 Hz, 2H), 6.89 (d, J = 7.5 Hz, 1 H), 3.10 (p, J = 3.7 Hz, 4H). 13 C NMR (151 MHz, DMSO-d6) d 154.7, 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] 391.1320, found: m / z 391.1322. + (C 22 H 20 BrN4O): m / z 435.0815, found: m / z 435.0819.

[0043]

[0044] 3.11 (dq, J = 8.5, 5.7, 4.7 Hz, 4H). 13 C NMR (151 MHz, DMSO-d6) d 154.8, 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] 391.1320, found: m / z 391.1322. + (C 23 H 20F3N4O): m / z 425.1584, found: m / z 425.1585. MS-ESI: calculated for [M+H] + (C 23 H 20 F3N4O): m / z 425.1584, found: m / z 425.1585.

[0045]

[0046] 13 C NMR (151 MHz, DMSO-d6) δ 158.5, 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: calculated for [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 = 7.5 Hz, 1H), 3.10 (tt, J = 6.9, 4.1 Hz, 4H), 2.24 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 154.8, 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 / z 371.1889.

[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) δ 154.8, 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 / z 385.2046.

[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) δ 154.8, 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 (151 MHz, DMSO-d6) δ 154.9, 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] 387.1816, found: m / z 387.1839. + (C 23 H 23 N4O2): m / z 401.1972, found: m / z 401.1999.

[0055]

[0056] 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). 13 C NMR (151 MHz, DMSO-d6) δ 154.9, 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] 387.1816, found: m / z 387.1839. + (C 24 H 25 N4O2): m / z 401.1972, found: m / z 401.1999.

[0057]

[0058] calculated for [M+H] 401.1972, found: m / z 401.1999. + (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] C32H26F3NO5Na, 577.1707, found: m / z 577.1709. + (C 36 H 36 BBrN4O5Na): m / z 717.1854, 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] C32H26F3NO5Na, 577.1707, found: m / z 577.1709. + (C 37 H 36 BF3N4O5Na): m / z 685.2804, found: m / z 685.2805.

[0067]

[0068] 133.1, 129.2, 128.5, 124.8, 124.7, 122.3, 120.4, 120.3, 119.7, 118.6, 116.5, 115.8, 115.6, 115.2, 84.2, 69.5, 33.2, 32.7, 25.1. MS-ESI: calculated for [M+H] C32H26F3NO5, 613.1753, found: m / z 613.1754. + (C 36 H 37 BFN4O5): m / z 635.2836, found: m / z 635.2837.

[0069] Experimental Example 1

[0070] Detection of transmembrane transport activity of compounds

[0071] Ion transport based on HPTS vesicle fluorescence experiments

[0072] Take 1 mL of dissolved in chloroform solution of egg yolk phospholipid (EYPC, 25 mg / mL) in a round bottom flask, remove chloroform at 35 °C under reduced pressure, dry overnight under vacuum. Add 1 mL of HTPS (0.1 mM, pH = 7.0, 100 mM NaCl, HEPES 10 mM) buffer solution containing pH-sensitive to the round bottom flask, and shake hydration at 37 °C for 2 hours. The resulting solution was placed in liquid nitrogen for 1 minute and then heated in a water bath at room temperature for 2 minutes, after 10 freeze-thaw cycles, the suspension was extruded through a polycarbonate membrane with a pore size of 100 nm to obtain a uniform size of vesicle (LUV) suspension, and finally the LUV suspension was dialyzed through a dialysis membrane to remove the fluorescent dye not coated in the LUV suspension to produce a lipid concentration of 6.5 mM LUV.

[0073] Add 30 μL of HPTS-coated LUV suspension 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 change of fluorescence intensity of the solution at 510 nm emission wavelength was detected by fluorescence spectrophotometer under 403 nm and 460 nm dual-wavelength excitation for 300 seconds. The final transport curve was obtained by calculating the ratio value of I 460 / I 403 , and the ratio value of I 460 / I 403 was normalized by equation (1) after adding Triton X-100.

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

[0075] where 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 Hill (S2) fitting of ion channel concentration to ion transport activity R curve obtained EC 50 value and Hill coefficient n.

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

[0078] As Figure 1As shown, HPTS experiments revealed that the ion transport activity of CR was affected by different substituents. C-Cl exhibited the highest activity; after background correction, C-Cl showed 93% ion transport activity at a concentration of 2 μM. The corresponding EC values ​​were obtained by measuring the transport activity of C-Cl at different concentrations. 50 The value is 0.21 μM ( Figure 2 ).

[0079] Figure 3 This explains how changing the type of alkali metal ions (M) in the external membrane buffer solution... + =Li + Na + ,K + ,Rb + Cs + When the ion transport activity of C-Cl remains essentially unchanged, it indicates that alkali metal ions do not participate in the ion transport process. Combined with molecular structural characteristics, Na can be ruled out. + / OH - Cotransport and Na + / H + Antitransport is the primary ion transport mechanism. Therefore, the main mechanism of C-Cl-promoted ion transport is Cl... - / H + Cotransport or Cl - / OH - Reverse transfer.

[0080] In the HPTS assay, the highly active proton transporter gramicidin (GA) was further used to compare Cl... - and H + The ion transport rate between them. And through highly active K + Selective transporter—valinemycin (VA) compared to Cl - and OH - Ion transport rate.

[0081] like Figure 4 As shown in figure a, C-Cl (0.21 μM) exhibits almost the same ion transport activity in the presence and absence of GA (100 pM), indicating that H + Or OH - The transmission speed is higher than Cl - quick. Figure 4 b indicates that VA and C-Cl (0.21 μM) did not show a synergistic effect, suggesting that Cl... - Transmission speed is faster than OH - Quickly. Combining the results of the two experiments, we can conclude that: H + / Cl - Co-directional transport is the primary transport mechanism, and H +The transfer speed is higher than Cl - Quick, Cl - The transport rate is higher than that of OH - quick.

[0082] Ion transport based on SPQ vesicle fluorescence experiments

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

[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 vesicle. After adding different concentrations of channel molecules with stirring, the fluorescence intensity change at the emission wavelength of 430 nm was detected using a fluorescence spectrophotometer at an excitation wavelength of 360 nm for 300 seconds. The final transport curve was obtained by calculating I... 460 / I 403 The ratio value was normalized using Equation (1) above after adding Triton X-100. The curve of ion channel concentration versus ion transport activity R was fitted using Equation (2) above to obtain EC. 50 Value and Hill coefficient n.

[0085] like Figure 5 As shown, when C-Cl was added to the LUV of the chloride ion-sensitive dye SPQ, a concentration-dependent quenching of SPQ fluorescence intensity was observed, EC 50 A value as low as 0.07 μM indicates that C-Cl efficiently promotes Cl- - Ion transmembrane transport, and the trend of chloride ion transport activity is consistent with that in HPTS assays.

[0086] Planar lipid bilayer experiments demonstrate that compounds are transported via an adaptive channel mechanism.

[0087] Measurement of chloride ion conductance (γ): 20 μL of DiPhyPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine, 10 mg / mL) chloroform solution was taken in a sample vial and blown dry with nitrogen and redissolved in 8 μL of n-decane. 0.5 μL of the lipid containing n-decane solution was injected into the well of a Delrin cup (Warner Instruments, Hamden, CT) (diameter = 200 μm) and N2 was blown to remove the n-decane. In patch clamp experiments, the chamber (cis) and Delrin cup (trans) were filled with KCl solution (1.0 M, 1.0 mL). Ag-AgCl electrodes were 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 well of the n-decane pretreated Delrin cup. 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 clamp 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 obtain the chloride conductance (γ). Measurement of chloride ion conductance (γ): 20 μL of DiPhyPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine, 10 mg / mL) chloroform solution was taken in a sample vial and blown dry with nitrogen and redissolved in 8 μL of n-decane. 0.5 μL of the lipid containing n-decane solution was injected into the well of a Delrin cup (Warner Instruments, Hamden, CT) (diameter = 200 μm) and N2 was blown to remove the n-decane. In patch clamp experiments, the chamber (cis) and Delrin cup (trans) were filled with KCl solution (1.0 M, 1.0 mL). Ag-AgCl electrodes were 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 well of the n-decane pretreated Delrin cup. 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 clamp 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 obtain the chloride conductance (γ).

[0088] Measurement of H + / Cl - ion transport selectivity: 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 N2 to form a thin film, which was redissolved in 50 μL of n-decane to produce a working solution with a channel to GMO molar ratio of 1 / 10 6 Measurement of H + / Cl - selectivity, defined as the ratio of permeability of the two ions (P H+ / P Cl- ), was obtained by fitting the I-V curves using the following simplified Goldman-Hodgkin-Katz equation (3).​

[0089] 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)

[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"1mol"1); T is the temperature in Kelvin (298 K); F is the Faraday constant (96485 C mol"1); and P is the permeability of the ion channel.

[0092] As Figure 6 shown, C-CI mediates the transmembrane transport of H + / Cl - through a channel mechanism rather than a carrier mechanism. By linearly fitting the current-voltage (I-V) plot, the Cl - conductance (γ Cl- ) of C-CI was determined to be 15.6 ± 0.1 ps. In addition, the H + / Cl - transport selectivity (P H+ / P Cl- ) of C-CI was calculated to be 7.7, further confirming that H + is transported faster than Cl - .

[0093] Experimental Example 2

[0094] Active oxygen stimulates release of active channel molecule

[0095] As Figure 7 shown in a, the introduction of a phenylboronic acid group in response to active oxygen significantly reduces ion transport activity. ROS-C-CI only requires 5 equivalents of H2O2 stimulation at low concentrations to restore 90% of the transmembrane transport activity. High performance liquid chromatography was used to assess the ability of ROS-C-CI to release the active channel molecule C-CI in the presence of H2O2, as Figure 7As shown in Figure b, after incubation of ROS-C-Cl (100 μM) with different concentrations of H202 for 30 min, the HPLC spectrum of ROS-C-Cl changed significantly. A new retention peak corresponding to the parent channel-forming unit C-Cl appeared, while the signal of ROS-C-Cl decreased in a concentration-dependent manner. After treatment with 5 equivalents of H202 for 30 min, 93% of ROS-C-Cl was converted to C-Cl.

[0096] Experimental Example 3

[0097] Anti-cancer activity and anti-cancer mechanism of the compounds

[0098] Anti-cancer activity and selectivity

[0099] Each well of a 96-well plate was seeded with ~10 4 breast cancer cells (MCF-7) and normal breast cells (MCF-10A), respectively, and incubated in a 5% CO2, 37 °C incubator overnight. The samples were dissolved in DMSO to prepare a series of solutions with different concentrations, which were then added to the 96-well plates containing the cells and incubated at 37 °C for 48 h. 20 μL of MTT solution was added to the culture medium, and the fluorescence of the solution was detected by a microplate reader to determine the cell activity and calculate the anti-cancer activity of the channel molecules on MCF-7 and their safety. 50 The IC50 values and selectivity indices of ROS-C-R on MCF-7 cells and MCF-10A cells are summarized in Table 1.

[0100] Table 1. IC50 values and selectivity indices of ROS-C-R on human MCF-7 cells and MCF-10A cells 50

[0101]

[0102] All four ROS-C-Rs showed good anti-cancer activity. The most active molecule, ROS-C-R, had an anti-breast cancer activity similar to that of paclitaxel (IC 50 = 2.8 μM). ROS-C-Cl showed excellent specificity for breast cancer cells, with 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 for detecting apoptosis

[0104] Each well of a 6-well plate was seeded with ~10 5 ​MCF-7 cells. After incubation at 37°C in a 5% CO2 incubator for 12 hours, the cells were treated with different concentrations of ROS-C-Cl (0, 5, 10 μM) for 36 hours. After removing the culture medium and washing the cells with PBS, the cells were trypsinized and collected, and 5 μL of FITC-Annexin-V and 10 μL of PI solution were added. The mixture was incubated at room temperature for 15 minutes in the dark, and the apoptosis of the tumor cells was detected by flow cytometry. As shown in Figure 2a, the percentage of apoptotic cancer cells increased from 2.4% to 15.7% as the concentration of ROS-C-Cl increased from 0 to 10 μM, confirming the ability of ROS-C-Cl to induce apoptosis in MCF-7 cells. Figure 8

[0105] Changes in mitochondrial membrane potential and ROS content

[0106] In each well of a 6-well plate, ~10 5 MCF-7 cells were seeded. After incubation at 37°C in a 5% CO2 incubator for 12 hours, the cells were treated with different concentrations of ROS-C-Cl (0, 5, 10 μM) for 24 hours. After removing the culture medium and washing the cells with PBS, the cells were stained with 1.0 mL of 1 x JC-1 staining working solution for 20 minutes, washed twice with 1 x JC-1 staining buffer, and then 1 mL of DMEM was added. Fluorescence images were obtained 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, while when the mitochondrial membrane is depolarized, the aggregates formed will depolymerize, emitting green fluorescence. The experimental results are shown in Figure 2b. ROS-C-Cl can cause an increase in green fluorescence and a decrease in red fluorescence in MCF-7 cells in a concentration-dependent manner, indicating that ROS-C-Cl can cause depolarization of the mitochondrial membrane potential. Figure 9

[0107] In each well of a 6-well plate, ~10 5 MCF-7 cells were seeded. After incubation at 37°C in a 5% CO2 incubator for 12 hours, the cells were treated with different concentrations of ROS-C-Cl (0, 5, 10 μM) for 6 hours. After removing the culture medium and washing the cells with PBS, the cells were trypsinized and collected, and then stained with 1.0 mL of reactive oxygen probe DCFH-DA staining working solution for 20 minutes. The relative ROS level in the cells was detected by flow cytometry. The experimental results are shown in Figure 2c. ROS-C-Cl can cause an increase in ROS content in MCF-7 cells in a concentration-dependent manner. Figure 9

[0108] Cytochrome c release study

[0109] MCF-7 cells were seeded at a density of 1 x 10 6 ​​​Cells were transplanted at a density of 10 cells / well onto glass coverslips 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 incubated with 0.5% Triton-X at 4°C for 10 minutes. After blocking the cells with 10% (v / v) FBS, they were stained overnight with cytochrome c antibody at 4°C. After staining, the cells were washed with PBS and then incubated with fluorescent secondary antibody. Finally, the cell boundaries 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. Figure 10 As shown, since elevated ROS levels cause changes in mitochondrial permeability, a significant enhancement of green fluorescence was observed after ROS-C-Cl treatment of cells, confirming the release of cytochrome c.

[0110] Apoptosis-related protein expression detection

[0111] MCF-7 cells were stored at a cell density of 1×10⁶. 6 Cells were seeded in 6-well plates and cultured for 24 h. After incubation with ROS-C-Cl (0, 5, 10, 20 μM) for 48 h, MCF-7 cells were lysed with RIPA buffer containing protease and phosphatase inhibitors (PMSF) for 20 min. Cell proteins were collected by centrifugation at 12,000 rpm for 10 min at 4 °C. Protein concentration was then determined using a BCA assay kit. An equal volume of protein (20 μg) was denatured in SDS sample buffer at 95 °C for 5 min, followed by separation using 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 h to prevent nonspecific binding. Then, it was incubated overnight at 4 °C with antibodies against apoptosis and autophagy-related proteins. Wash three times with TBST for 10 min each time, then incubate at 25°C for 2 h with horseradish peroxidase-labeled secondary antibody solution (Abcam) at room temperature, followed by washing with 1×TBST solution. Finally, develop color with ECL reagent, observe and photograph using an exposure meter.

[0112] like Figure 11As shown, using the above immunoblotting method to show the apoptosis-related proteins, treating MCF-7 cells with ROS-C-Cl for 48 hours caused a concentration-dependent downregulation of the anti-apoptotic proteins MCL1 and BCL2, indicating that ROS-C-Cl can promote MCF-7 cell apoptosis through the mitochondrial pathway. The observed upregulation of the pro-apoptotic proteins cleaved caspase9 and cleaved PARP further supports ROS-C-Cl-induced apoptosis, indicating that the apoptosis cascade is fully activated.

[0113] Artificial ion channels promote lysosomal pH and induce autophagy

[0114] In 6-well plates, ~10 5 MCF-7 cells were seeded in each well. After incubation at 37°C in a 5% CO2 incubator for 12 hours, different concentrations of ROS-C-Cl (0, 5, 10 μM) were added to treat the cells for 12 hours. After removing the culture medium and washing the cells with PBS, the cells were trypsinized without EDTA and the cell suspension was collected and stained with 1.0 mL of AO staining solution (15 μg / mL) for 20 minutes, and then washed twice with PBS and added with 1 mL of DMEM. The fluorescence images were obtained using a laser confocal microscope. As shown in Figure 12 , ROS-C-Cl can increase lysosomal pH and immunoblotting shows that the autophagy-related proteins LC3-II and p62 are upregulated, indicating that ROS-C-Cl can interfere with the autophagy process.

[0115] Unlike existing tubular cavities and non-covalent pegging artificial H + / Cl - channel construction strategies, the present application is based on a completely different molecular structure, which utilizes the structural characteristics of imidazole having both lone pair electrons and polar N-H bonds. The lone pair electrons and polar hydrogen are used as the recognition units of H + and Cl - , respectively. Then, by one-dimensional assembly of the ion recognition units through intermolecular hydrogen bonds, an artificial channel capable of synergistically transporting H + and Cl - is constructed, and the H + / Cl - ion transport selectivity is 7.7. In addition, unlike the existing artificial H + / Cl - channels that respond to external stimuli such as visible light, the present application introduces groups that respond to endogenous stimuli such as reactive oxygen species to the artificial H + / Cl - channel, so that it can only be activated under the stimulation of reactive oxygen species that are highly expressed only in cancer cells. After activation, ROS-C-Cl activates the Caspase 9 apoptosis pathway and disrupts the autophagy process, IC 50was 2.8 mM. Most importantly, by taking advantage of the inherent oxidative microenvironment of cancer cells and the H + / Cl - Enhanced oxidative stress caused by co-transport, ROS-C-Cl exhibited excellent selectivity in targeting breast cancer cells with a selectivity index of 10.2 compared to normal breast cells. This selectivity greatly exceeded that of the traditional chemotherapeutic drug paclitaxel by 20.2 times, highlighting the ROS-responsive artificial H + / Cl - ion channel as a great potential for cancer-specific therapy.

Claims

1. An active oxygen-responsive artificial ion channel, characterized by, The structural general formula is as follows: 。 2. The method of producing an active oxygen-responsive artificial ion channel according to claim 1, wherein The synthesis route is as follows: (1) 3-(3-nitrophenyl)propionic acid 1a and o-phenylenediamine 1b as raw materials, in the presence of phosphorus oxychloride, high temperature reaction at 120°C cyclization to generate 1c containing benzimidazole structure, then under the condition of palladium on carbon / hydrogen, reduction of nitro to amine 1d, and then reacted with different para-substituted isocyanate to generate urea structure, to prepare a class of artificial H + / Cl - ion channel C-R, wherein R = F, Cl, Br, CF3; (2) Activity oxygen response modification of channel molecules: first, 4-(hydroxymethyl) phenylboronic acid pinacol ester is reacted with p-nitrophenyl chloroformate to prepare intermediate 2c, and then the artificial H + / Cl - ion channel C-R is reacted with intermediate 2c in the presence of NaH to prepare a class of activity oxygen response type artificial H + / Cl - ion channel ROS-C-R, wherein R = F, Cl, Br, CF3.

3. The method for preparing a reactive oxygen species-responsive artificial ion channel according to claim 2, characterized in that, The synthesis route is as follows: 。 4. Use of the active oxygen-responsive artificial ion channel of claim 1 in the preparation of a drug for anti-cancer.

5. A pharmaceutical composition comprising the active oxygen-responsive artificial ion channel of claim 1 and a pharmaceutical adjuvant.

Images