A GSH-responsive artificial K + / X - Co-transport ion channel compounds, methods of making and anti-tumor applications thereof

Abstract

The application relates to a GSH-responsive artificial K + / X ‑ Co-transport ion channel compound, preparation method thereof and antitumor application. The compound takes a single peptide as a skeleton, introduces a crown ether potassium ion recognition unit, and promotes self-assembly in a lipid membrane through a hydrophobic alkyl chain; a 2,4-dinitrophenylsulfonyl group is further introduced at a tyrosine hydroxyl position of the single peptide, so that the compound is selectively released in a high glutathione microenvironment. The compound can self-assemble on a cell membrane to form a K + / X ‑ Co-transport artificial ion channel, disturbs the potassium ion and anion homeostasis in tumor cells, induces a decline in a mitochondrial membrane potential, accumulation of reactive oxygen species and glutathione depletion, and thus triggers tumor cell apoptosis. The compound has the advantages of clear structure, high selectivity, strong tumor microenvironment responsiveness and low toxic and side effects, and is suitable for the preparation of an antitumor drug or a related preparation.

Description

Technical Field

[0001] This invention belongs to the fields of artificial ion channels, chemical biology and anti-tumor drugs, specifically relating to an artificial potassium / anion cotransport ion channel compound with glutathione responsive properties, and also to the preparation method of the compound and its application in regulating cellular ion homeostasis, especially in anti-tumor therapy. Background Technology

[0002] Ion channels are important functional units on biological membranes that regulate the transmembrane transport of inorganic ions, playing a crucial role in maintaining intracellular and extracellular ion homeostasis, membrane potential stability, cell volume regulation, and signal transduction. Potassium ions and anions, as the most abundant ion pairs inside and outside the cell, directly influence the physiological state of the cell through their transmembrane distribution and dynamic balance.

[0003] Studies have shown that during malignant transformation and rapid proliferation, tumor cells undergo significant alterations in cell membrane permeability, ion channel expression, and ion homeostasis regulation mechanisms. Potassium and anion channels, in particular, play crucial roles in cell cycle regulation, migration and invasion, and apoptosis escape in tumor cells. Artificially disrupting the potassium and anion balance within tumor cells may induce functional disorders and initiate programmed cell death.

[0004] In recent years, artificial ion channels, as molecular systems that mimic the function of natural ion channels, have attracted widespread attention. Artificial ion channels typically self-assemble into transmembrane channel structures in a lipid membrane environment through the synergistic effect of molecular recognition units and hydrophobic frameworks, thereby achieving selective transport of specific ions. Compared to natural ion channels, artificial ion channels offer advantages such as designable structures and tunable functions, and have significant application potential in basic research and biomedical fields.

[0005] However, existing artificial ion channel technology still has the following shortcomings:

[0006] (1) Most artificial ion channels are only for a single ion type and it is difficult to achieve multi-ion synergistic transport;

[0007] (2) Some artificial ion channels have insufficient ion selectivity, which can easily disrupt the ion homeostasis of normal cells;

[0008] (3) Lack of responsiveness to the tumor microenvironment may lead to non-specific toxicity in normal tissues.

[0009] Glutathione is a widely distributed reducing tripeptide in cells, playing a crucial role in maintaining intracellular redox homeostasis. Numerous studies have found that glutathione levels in tumor cells are typically significantly higher than in normal cells, providing an important basis for constructing tumor-selectively responsive functional molecules. By introducing glutathione-responsive groups, functional molecules can be selectively activated in tumor cells, which helps improve therapeutic selectivity and reduce side effects.

[0010] Therefore, developing an artificial ion channel system that can be activated in the tumor high-glutathione microenvironment and synergistically disrupt potassium and anion homeostasis is a technical problem that urgently needs to be solved in the current research field of artificial ion channels and anti-tumor therapy. Summary of the Invention

[0011] The purpose of this invention is to overcome the shortcomings of existing artificial ion channels in terms of selectivity, controllability, and anti-tumor applications, and to provide an artificial K+ channel with a well-defined structure that can self-assemble in a lipid membrane and exhibits glutathione-responsive properties. + / X - (including F) - Cl - ,Br - and I - This study provides information on cotransport ion channel compounds, their preparation methods, and their applications in antitumor therapy.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] 1. By using a single peptide as the molecular backbone and introducing crown ether structural units with potassium ion recognition capabilities, the compound exhibits high binding selectivity for potassium ions;

[0014] 2. Introducing hydrophobic alkyl side chains into the monopeptide backbone enables the compound to embed into the lipid bilayer and undergo self-assembly in the membrane environment;

[0015] 3. Introducing glutathione responsive groups at specific sites of a single peptide causes the compound to undergo structural transformation in a high glutathione environment, releasing a molecular structure with channel activity;

[0016] 4. After self-assembly in a membrane environment, the compound forms an artificial ion channel capable of synergistically transporting potassium ions and anions, thereby disrupting intracellular ion homeostasis.

[0017] Furthermore, a GSH-responsive artificial K + / X -Cotransport ion channel compounds include: a monopeptide backbone; a crown ether potassium ion recognition unit covalently linked to the monopeptide backbone; 2,4-dinitrobenzenesulfonyl (DNS) as an upregulating glutathione (GSH) responsive group; and at least one hydrophobic alkyl side chain. The compounds are capable of self-assembling in lipid membranes to form transmembrane ion channels.

[0018] Furthermore, the crown ether potassium ion recognition and transport unit is benzo-15-crown-5, benzo-15-crown-6, benzo-15-crown-7, benzo-15-crown-8 or its substitutes.

[0019] Furthermore, the monopeptide backbone contains tyrosine residues.

[0020] Furthermore, the hydroxyl position of the tyrosine residue is attached to a glutathione responsive group.

[0021] Furthermore, the glutathione responsive group is a 2,4-dinitrobenzenesulfonyl group.

[0022] Furthermore, the hydrophobic alkyl side chain is a C8-C12 straight-chain or branched alkyl group.

[0023] Furthermore, the GSH-responsive artificial K + / X - The structural formula of the cotransport ion channel compound (5Yn-DNS) is as follows:

[0024] Where n = 8~12.

[0025] Furthermore, a GSH-responsive artificial K + / X - The preparation method of cotransport ion channel compounds includes the following steps:

[0026] 1) Synthesize carboxyl-substituted benzocrown ether intermediates;

[0027] 2) The benzocrown ether intermediate is subjected to an amidation reaction with a single peptide backbone containing a hydrophobic alkyl chain to obtain the artificial ion channel parent compound;

[0028] 3) Optionally, the parent compound is reacted with a dinitrobenzenesulfonyl halide to introduce a glutathione responsive group.

[0029] Application: The use of the artificial ion channel compound in the preparation of formulations for regulating cellular ion homeostasis.

[0030] Application: The use of the aforementioned artificial ion channel compound in the preparation of antitumor drugs, which disrupt intracellular K+ in tumor cells. + Anion homeostasis induces apoptosis.

[0031] The advantages of this invention are:

[0032] (1) Selective recognition of potassium ions is achieved through crown ether structure, thereby improving the specificity of ion transport;

[0033] (2) To achieve the synergistic transport of potassium ions and anions, thereby enhancing the ability to interfere with the ion homeostasis of tumor cells;

[0034] (3) Introducing a glutathione response mechanism to preferentially activate the compound in tumor cells and improve therapeutic selectivity;

[0035] (4) It can simultaneously induce glutathione depletion and reactive oxygen species accumulation in cells, forming multiple pro-apoptotic effects;

[0036] (5) The compound structure can be designed and the preparation method is mature, and it has good application prospects. Attached Figure Description

[0037] Figure 1 (a)5Y8 transports K at different concentrations + Test results; (b) 5Y8 transport K + EC50 fitting plot; (c) 5Y10 transports K at different concentrations + Test results; (d) 5Y10 transport K + EC50 fitting plot. Detailed Implementation

[0038] To make the above-mentioned features and advantages of the present invention more apparent and understandable, specific embodiments are described below in detail. Unless otherwise specified, the methods of the present invention are conventional methods in the art.

[0039] Example 1

[0040] Preparation of carboxyl-substituted benzocrown ether intermediates

[0041] Starting with methyl 3,4-dihydroxybenzoate (1 mmol), an etherification reaction was carried out with tetraethylene glycol di-p-toluenesulfonate (1 mmol) under alkaline conditions (K₂CO₃, 2 mmol). The benzocrown ether skeleton was constructed in DMF at 85 °C to obtain a carboxyl-substituted benzo-15-crown-5 intermediate. After the reaction was complete, the reaction solution was extracted, washed, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain a white or off-white solid product. The results of 1H NMR and mass spectrometry characterization showed that the structure of the obtained product was consistent with the target intermediate, and the purity met the requirements for subsequent reactions. 1H NMR (400 MHz, Chloroform-d) δ 7.79 (dd, J = 8.4, 1.9 Hz, 4H), 7.37 - 7.30 (m, 4H), 4.17 - 4.13 (m,4H), 3.68 (td, J = 5.0, 1.8 Hz, 4H), 3.56 (d, J = 1.8 Hz, 8H), 2.44 (d, J =1.7 Hz, 6H), yield 86%.

[0042]

[0043] Synthesis of carboxyl-substituted benzocrown ethers

[0044] Example 2

[0045] Synthesis of single peptide backbone intermediates

[0046] Using N-tert-butoxycarbonyl-L-tyrosine (10 mmol) as a starting material and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) as a catalyst (15 mmol), amino acid residues were progressively introduced onto n-octylamine or n-decanamine (10 mmol). The reaction was carried out at room temperature for 18 hours in a DMF:DCM ratio of 1:1 (10 mL) solvent to construct a monopeptide backbone containing tyrosine residues and hydrophobic alkyl side chain amino acids. After peptide chain elongation, the monopeptide was released through deprotection and cleavage steps. The crude product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain the target monopeptide intermediate (yield 93%). Mass spectrometry analysis showed that its molecular weight was consistent with the theoretical value, indicating that the monopeptide backbone was successfully constructed.

[0047]

[0048] Single peptide backbone intermediate

[0049] Example 3

[0050] Preparation of artificial ion channel parent compound 5Y8

[0051] The product obtained in Example 1 (2 mmol) was added to a round-bottom flask, along with excess sodium hydroxide aqueous solution (1 mL of 1 M NaOH solution) and a small amount of ethanol (5 mL) as a co-solvent. The mixture was heated under reflux for 30 minutes. Dilute hydrochloric acid (2 M HCl) was carefully added dropwise under ice bath cooling and stirring to acidify to a strongly acidic state (pH < 2), at which point a large amount of white crown ether benzoate solid precipitated. Finally, the solid was collected by vacuum filtration, and the filter cake was washed several times with a small amount of ice water.

[0052] The compound BYn (1 mmol) from Example 2 was dissolved in dichloromethane (5 mL). A nitrogen balloon was attached to a round-bottom flask to ensure operation under nitrogen protection. The reaction mixture was placed in an ice bath. Trifluoroacetic acid (TFA, 10 mmol) was slowly added dropwise under stirring, controlling the addition rate to suppress exothermic reaction and bubble generation. After the addition was complete, the ice bath was removed, and the reaction system was stirred at room temperature for 12 hours. After the reaction was complete, the system was cooled to 0°C again, and excess TFA was slowly neutralized by adding saturated sodium bicarbonate solution to adjust to neutral (pH ≈ 7), while stirring to prevent emulsification. The organic phase was extracted with dichloromethane (4 × 60 mL), combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the target compound NH2-Tyr-Cn (yield: 93%) as a pale yellow solid.

[0053] The crown ether benzoate (2 mmol) was then dissolved in anhydrous dichloromethane (10 mL), and the condensing agent BOP (3 mmol) and the basic auxiliary agent DIEA (3 mmol) were added. The reaction was stirred for 24 h in an ice bath (0 °C). Subsequently, NH2-Tyr-Cn (1 mmol, 1 mL aqueous solution) was slowly added dropwise, and the reaction was continued for 3 h at room temperature. After the reaction was completed, the system was washed with water, the organic phase was separated and concentrated under reduced pressure, and purified by column chromatography to obtain the target artificial ion channel parent compound 5Yn. Characterization results showed that the crown ether recognition unit was successfully covalently linked to the single peptide backbone. 1 H NMR (500 MHz, DMSO-d6) δ 9.13(s, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.96 (t, J = 5.7 Hz, 1H), 7.42 (dd, J =8.4, 2.1 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.11 - 7.08 (m, 2H), 6.97 (d, J =8.4 Hz, 1H), 6.64 - 6.59 (m, 2H), 4.53 (ddd, J = 10.1, 8.4, 5.0 Hz, 1H), 4.11- 4.05 (m, 4H), 3.78 (dt, J = 6.8, 3.2 Hz, 4H), 3.63 - 3.60 (m, 8H), 3.09 -3.00 (m, 2H), 2.97 - 2.79 (m, 2H), 1.37 (d, J = 7.3 Hz, 2H), 1.23 (d, J = 6.3Hz, 10H), 0.86 - 0.83 (m, 3H). 13C NMR (126 MHz, DMSO-d6) δ 171.39, 165.60,155.71, 151.06, 147.70, 130.12, 128.51, 126.62, 121.07, 114.82, 112.75,112.25, 70.53, 69.75, 69.66, 68.79, 68.66, 68.53, 68.25, 55.33, 38.52, 36.78,31.28, 29.06, 28.75, 28.71, 26.35, 22.14, 14.01. MS-ESI: calculated for [M+H]+(C32H47N2O8): m / z 587.3327, found: m / z 587.3332. (5Y8)Yield 64%.

[0054]

[0055] Synthesis route of 5Yn (n = 8, 10)

[0056] Example 4

[0057] Preparation of GSH-responsive artificial ion channel compound 5Y8-DNS

[0058] The 5Y8 (1 mmol) obtained in Example 3 was dissolved in anhydrous dichloromethane (10 mL), and 2,4-dinitrobenzenesulfonyl chloride (2 mmol) was added at pH = 8. The reaction temperature was controlled at 6 °C, and the reaction was stirred in the dark for 8 h. After the reaction was completed, the target GSH-responsive artificial ion channel compound 5Y8-DNS was obtained by quenching, extraction, washing, and purification steps, with a yield of 52%. NMR and mass spectrometry analysis showed that the dinitrobenzenesulfonyl group was successfully introduced into the tyrosine hydroxyl site. 1¹H NMR (500 MHz, DMSO-d₆) δ 9.08 (d, J = 2.2 Hz, 1H), 8.50 - 8.48 (m, 1H), 8.42 (d, J = 8.4 Hz, 1H), 8.08 (d, J = 8.7 Hz, 1H), 8.03 (t, J = 5.7 Hz, 1H), 7.41 - 7.39 (m, 1H), 7.38 - 7.36 (m, 2H), 7.35 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.7, 1.8 Hz, 2H), 6.97 (d, J = 8.4 Hz, 1H), 4.60 (td, J = 9.2, 5.0 Hz, 1H), 4.08 (dd, J = 5.6, 3.5 Hz, 4H), 3.78 - 3.75 (m, 4H), 3.61 (d, J = 2.1 Hz, 8H), 3.06 - 2.98 (m, 4H), 1.34 (d, J = 7.6 Hz, 2H), 1.23 - 1.20 (m, 10H), 0.85 - 0.82 (m, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 170.85, 165.66, 151.40, 148.08, 147.73, 147.05, 138.94, 133.41, 131.13, 130.86, 127.28, 121.48, 121.08, 112.73, 70.50, 69.73, 69.63, 68.77, 68.64, 68.55, 68.25, 54.60, 31.24, 28.99, 28.70, 28.66, 26.32, 22.09, 13.95. MS-ESI: calculated for [M+H]⁺ (C₃₈H₄₉N₄O₁₄S): m / z 817.2961, found: m / z 817.2950. The synthesis of 5Y10-DNS refers to that of 5Y8-DNS. 1HNMR (400 MHz, DMSO-d6) δ 9.07 (d, J = 2.3 Hz, 1H), 8.50 (dd, J = 8.7, 2.4 Hz,1H), 8.40 (dd, J = 8.6, 3.6 Hz, 1H), 8.09 (dd, J = 8.7, 2.1 Hz, 1H), 8.01 (t,J = 5.7 Hz, 1H), 7.42 - 7.35 (m, 4H), 7.07 (d, J = 8.3 Hz, 2H), 6.96 (d, J =8.3 Hz, 1H), 4.61 (td, J = 9.2, 5.0 Hz, 1H), 4.08 (hept, J = 4.9, 4.2 Hz,4H), 3.77 (q, J = 4.0 Hz, 4H), 3.61 (s, 8H), 3.01 (tdd, J = 13.6, 7.5, 3.9Hz, 4H), 1.36 (q, J = 6.9 Hz, 2H), 1.22 (s, 14H), 0.84 (t, J = 6.7 Hz, 3H).13C NMR (101 MHz, DMSO-d6) δ 170.79, 165.62, 151.34, 151.15, 148.04, 147.73,147.01, 138.89, 133.36, 131.08, 130.88, 127.24, 126.44, 121.42, 121.02,112.78, 112.26, 70.48, 69.73, 69.63, 68.76, 68.59, 68.27, 54.57, 44.12,38.51, 36.77, 31.26, 28.97, 28.92, 28.70, 28.67, 26.27, 22.05, 13.91. MS-ESI: calculated for [M+H]+ (C40H53N4O14S): m / z 845.3274, found: m / z 845.3265.

[0059] Example 5

[0060] Testing the transmembrane ion transport performance of artificial ion channels in liposome systems

[0061] A liposome model mimicking the cell membrane was prepared using a phospholipid self-assembly method, and the artificial ion channel compound was embedded in the lipid bilayer. Changes in potassium and anion concentrations inside and outside the liposome were monitored using ion-selective electrodes or fluorescent probes. Experimental results showed that in the presence of the compound, the potassium concentration in the liposome system... + Significant transmembrane transport occurs, accompanied by anions (including F). - Cl - ,Br - and I - The compound exhibited co-migration of K+; compared to the control group, the ion flux was significantly enhanced, indicating that the compound can self-assemble into a functional K+ in the membrane environment. + / X - Common transfer channel ( Figure 1 ).

[0062] Example 6

[0063] GSH Response Activation Behavior Verification Experiment

[0064] The 5Y8-DNS obtained in Example 4 was incubated in low-concentration (1 μmol / mL) and high-concentration (5 μmol / mL) glutathione solutions, respectively, and its structural changes were monitored by UV-Vis absorption spectroscopy or fluorescence spectroscopy. The results showed that under high GSH conditions, the dinitrobenzenesulfonyl group underwent a cleavage reaction, releasing the active artificial ion channel parent compound; while under low GSH conditions, the compound remained stable, indicating that it has obvious GSH-responsive characteristics.

[0065] Example 7

[0066] In vitro antitumor activity assay of artificial ion channel compounds

[0067] Multiple tumor cell lines were selected as models, and different concentrations of artificial ion channel compounds were co-incubated with the cells for a certain period of time. The effect on cell proliferation was evaluated using cell viability assay. The results showed (Table 1) that the compounds had a significant inhibitory effect on tumor cell proliferation in a concentration-dependent manner; compared with compounds without GSH responsive groups, the GSH-responsive compounds had a more significant inhibitory effect on tumor cells.

[0068] Table 1. Effects of channel molecules on IC50 in different cell lines 50 value

[0069]

[0070] Example 8

[0071] Validation of the mechanism of inducing tumor cell apoptosis

[0072] The mechanism of action of the compound was analyzed by detecting changes in intracellular reactive oxygen species levels, mitochondrial membrane potential, and glutathione content. Experimental results showed that the artificial ion channel compound could significantly disrupt intracellular K+ in tumor cells. + It disrupts anion homeostasis, leading to mitochondrial dysfunction, ROS accumulation, and GSH depletion, ultimately inducing apoptosis.

[0073] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A GSH-responsive artificial K + / X - Cotransport ion channel compounds, characterized in that, The compound comprises: a monopeptide backbone; a crown ether potassium ion recognition unit covalently linked to the monopeptide backbone; 2,4-dinitrobenzenesulfonyl DNS as an upregulating glutathione GSH responsive group; at least one hydrophobic alkyl side chain; wherein the compound is capable of self-assembling in a lipid membrane to form a transmembrane ion channel.

2. The compound according to claim 1, characterized in that, The crown ether potassium ion recognition unit is benzo-15-crown-5, benzo-15-crown-6, benzo-15-crown-7, benzo-15-crown-8 or its substitutes.

3. The compound according to claim 1, characterized in that, The monopeptide backbone contains tyrosine residues, and the hydroxyl positions of the tyrosine residues are linked to glutathione responsive groups.

4. The compound according to claim 3, characterized in that, The glutathione responsive group is a 2,4-dinitrobenzenesulfonyl group.

5. The compound according to claim 1, characterized in that, The hydrophobic alkyl side chain is a C8-C12 straight-chain or branched alkyl group.

6. The compound according to claim 1, characterized in that, The structural formula of the compound is as follows: Where n = 8~12.

7. The GSH-responsive artificial K according to any one of claims 1-6 + / X - A method for preparing cotransport ion channel compounds, characterized in that, Includes the following steps: 1) Synthesize carboxyl-substituted benzocrown ether intermediates; 2) The benzocrown ether intermediate is subjected to an amidation reaction with a single peptide backbone containing a hydrophobic alkyl chain to obtain the artificial ion channel parent compound; 3) The parent compound is reacted with dinitrobenzenesulfonyl halide to introduce a glutathione responsive group.

8. The GSH-responsive artificial K according to any one of claims 1-6 + / X - Application of cotransport ion channel compounds in the preparation of formulations for regulating cellular ion homeostasis.

9. The GSH-responsive artificial K according to any one of claims 1-6 + / X - Application of cotransport ion channel compounds in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The antitumor drug works by disrupting the intracellular K+ of tumor cells. + Anion homeostasis induces apoptosis.

Images