An artificial potassium ion transporter with anti-hepatic fibrosis activity and its preparation method and application

Abstract

The present invention discloses an artificial potassium ion transporter with anti-liver fibrosis activity, its preparation method and application, wherein the compound uses crown ether as the potassium ion transport unit and asiatic acid as the cell membrane anchoring unit, and 10 artificial potassium ion transporters are constructed by connecting the crown ether with carbon chains of different lengths. The present invention is based on the concept of molecular machines, according to the various natural K + Given the potential for HSC activation induced by dysfunction of related ion pumps and channels, a novel class of artificial potassium ion transporters has been designed and synthesized based on the anti-inflammatory and antioxidant properties of asiatic acid. The synthetic route presented herein is environmentally friendly, simple and efficient, and utilizes readily available raw materials, making it applicable to the synthesis of similar compounds. The compounds presented herein are highly selective and active artificial ion transporters, potentially useful in anti-fibrosis treatments. This approach aims to provide novel and effective candidate compounds for the treatment of liver fibrosis and to prepare reliable anti-liver fibrosis compounds.

Description

Technical Field

[0001] The invention relates to an artificial potassium ion transporter with anti-liver fibrosis activity and a preparation method and application thereof, belonging to the field of medicine. Background Art

[0002] Liver fibrosis is the result of many chronic liver diseases, including chronic hepatitis B and C virus infection, alcohol abuse, biliary obstruction, autoimmune diseases, hereditary hemochromatosis, and non-alcoholic fatty liver disease (NAFLD). Liver fibrosis can further develop into cirrhosis, liver cancer, and other diseases, ultimately endangering life. Early treatment of liver fibrosis mainly removes potential damaging stimuli, including etiological treatment such as antiviral therapy and treatment of the primary disease. Unfortunately, with the exception of some traditional Chinese medicine compound preparations, there are currently no marketed chemical drugs or biological agents for the treatment of liver fibrosis. Developing a new class of reliable anti-fibrosis drugs is an urgent problem that needs to be solved.

[0003] Hepatic stellate cells are fibroblasts found in the liver, and their abnormal proliferation and excessive differentiation are key processes in the pathogenesis of fibrosis. Numerous evidence indicates that dysfunction of natural ion channels or enzymes related to intracellular K+ concentrations, such as Na+, K+-ATPase, and BK channels, can significantly affect fibroblast activation and proliferation.

[0004] Na+, K+-ATPase is ubiquitously distributed on the cell membrane. Its primary function, under the action of ATP, is to pump three Na+ ions out of the cell against a concentration gradient and two K+ ions into the cell against a concentration gradient. Researchers have found that cardiotonic steroids, a potent inhibitor of the Na+-K+ pump, can significantly reduce K+ concentrations in various fibroblasts, triggering protein kinase A activation and reducing TGF-β-induced fibrosis. Ouabain, another sodium pump inhibitor, can increase the [Na+]i / [K+]i ratio in fibroblasts, thereby reducing the expression of TGF-βR2, a key TGF-β receptor. By inhibiting the TGF-β1 / Smad2 / 3 signaling pathway, it reduces fibroblast activation and extracellular matrix production, reversing the progression of liver fibrosis. Upregulating Na+-K+ pump activity can significantly enhance TGF-β signaling.

[0005] Furthermore, the functional loss and downregulation of BK channels, another type of potassium ion channel that causes K+ efflux, have also been shown to be associated with the activation of HSCs. First, activation of BK channels by various specific agonists, such as rottlerin, can directly lead to significant downregulation of multiple pro-fibrotic signaling pathways in vitro and in vivo. In vivo experiments with CCl4-induced liver fibrosis have also shown that BK channels can inhibit the function of HSCs. Second, the opening of BK channels can promote the degradation and endocytosis of TGF-β receptors, indirectly promoting the degradation of the TGF-β1 / Smad signaling pathway, thereby reducing the activation and differentiation of fibroblasts. Finally, researchers have also found that the deletion and knockout of the BK gene can significantly aggravate tissue fibrosis.

[0006] Therefore, it is not difficult to conclude that regulating the intracellular potassium ion concentration to antagonize the overactivation of the Na+-K+ pump and the loss or functional downregulation of BK channels is a new direction for the treatment of fibrosis. However, some ion pump inhibitors or BK channel agonists have problems such as a narrow therapeutic window and easy development of drug resistance, which limits their further development and application, while there is no reliable treatment for BK channel loss. The chemical synthesis of artificial potassium ion transporters with transmembrane transport activity is expected to provide a potential solution for the treatment of related diseases caused by potassium ion transport defects. Artificial potassium ion transporters can utilize the difference in K+ concentration inside and outside the membrane to achieve transmembrane transport of K+. However, how to construct artificial potassium ion transporters with high transmembrane transport activity and high potassium / sodium ion selectivity and use them to treat liver fibrosis still needs breakthroughs.

[0007] As of the end of 2021, more than 10 drugs for the treatment of liver fibrosis have entered, completed or terminated Phase III clinical trials. Among them, only the farnesoid FXR receptor agonist obeticholic acid (OCA) has completed Phase III trials. However, the FDA believes that the benefits of this drug treatment do not outweigh the potential risks, and the treatment group has severe itching (51%). Therefore, OCA has not yet been approved for the treatment of liver fibrosis. Phase III clinical trials of some drugs such as Cenicriviroc, Elafibranor, and Selonsertib were terminated early because the mid-term assessment did not meet the endpoint indicators. Most drugs targeting other targets such as TGF-β have systemic metabolic abnormalities, are prone to drug resistance, and cannot take into account the chronic process of metabolic liver disease. If liver fibrosis develops to the terminal stage, liver transplantation will be the only treatment method, but problems such as insufficient organs available for transplantation and high medical costs will bring huge uncertainty and economic burden to patients' treatment. Therefore, effective anti-fibrosis treatment has become an urgent issue. To address the above issues, the present invention intends to develop a novel, less resistant, and highly safe artificial potassium ion transporter based on the regulation of changes in intracellular K+ concentration on the pathogenesis of liver fibrosis, and apply it to anti-fibrosis treatment. Summary of the Invention

[0008] In view of the above problems existing in the prior art, the purpose of the present invention is to obtain an artificial potassium ion transporter with anti-hepatic fibrosis activity and its preparation method and application.

[0009] To achieve one of the above-mentioned objects of the invention, the general formula of the artificial potassium ion transporter with anti-hepatic fibrosis activity used in the present invention is as follows:

[0010]

[0011] Here, m is 1 or 2, and n is 1 to 5. That is, m = 1 represents 15-crown-5, and m = 2 represents 18-crown-6. n represents different carbon chain lengths, including 4, 6, 8, 10, and 12 carbon chain lengths.

[0012] Preferably, the compound uses crown ether as a potassium ion transport unit and asiatic acid as a cell membrane anchoring unit. Ten artificial potassium ion transporters are constructed by connecting the crown ether with carbon chains of different lengths, specifically: 5C4, 6C4, 5C6, 6C6, 5C8, 6C8, 5C10, 6C10, 5C12, and 6C12. The values ​​of m and n for different transporters are shown above, and the structural formula is as follows:

[0013]

[0014]

[0015] or their stereoisomers, tautomers, pharmaceutically acceptable salts. Specifically, they may be derivatives, analogs, stereoisomers, diastereomers, geometric isomers, polymorphs, solvates, cocrystals, intermediates, metabolites, prodrugs, or pharmaceutically acceptable salts and compositions thereof.

[0016] The synthetic route of the target compound provided by the present invention is as follows:

[0017]

[0018] The specific synthesis process is as follows: (1) reacting dibromoalkanes of different lengths with crown ether benzoic acid to obtain intermediate compounds XCn-Br (n = 4, 6, 8, 10, 12; X = 5 or 6) (2) alkylating XCn-Br in (1) with Centella asiatica acid to obtain the target compound XCn.

[0019] Another object of the present invention is to provide an application of the above-mentioned compound transporter in detecting the potassium ion transmembrane transport activity and ion selectivity of the target compound, and further, in testing the anti-liver fibrosis activity of the target compound.

[0020] The compounds described in this invention exhibit excellent K+ transport activity. Studies based on pH-sensitive HPTS vesicle fluorescence demonstrate that the compounds possess excellent potassium ion transmembrane transport activity and selectivity. Self-quenching CF dye leakage experiments also reveal that the addition of the target compounds does not directly disrupt cell membrane integrity, demonstrating their fundamental properties as artificial ion transporters.

[0021] In view of the fact that the artificial potassium ion transporter provided by the present invention has good K+ transport activity and anti-liver fibrosis activity, the present invention also provides a pharmaceutical composition, which uses the above-mentioned artificial potassium ion transporter as an active ingredient.

[0022] Furthermore, in vitro and in vivo anti-hepatic fibrosis activity experiments have shown that this class of compounds exhibits good anti-hepatic fibrosis activity and safety, has potential for development as anti-hepatic fibrosis drugs, and can be used to prepare anti-hepatic fibrosis drugs. The compounds prepared by the present invention can inhibit the proliferation and differentiation of hepatic stellate cells (HSC-T6) activated by TGF-β1 and affect cell morphology. Therefore, the present invention also provides the use of the compounds in the preparation of drugs for treating and / or preventing anti-hepatic fibrosis. Specifically, the following further applications are provided:

[0023] The compound prepared by the present invention can reduce the expression of fibrosis markers α-SMA and Col I, thus providing application of the compound in drugs for reducing the expression of fibrosis markers α-SMA and Col I.

[0024] The compound prepared by the present invention can induce apoptosis and block cell cycle of hepatic stellate cells HSC-T6 activated by TGF-β1, thus providing the use of the compound in drugs for inhibiting proliferation and differentiation of hepatic stellate cells HSC-T6 activated by TGF-β1.

[0025] The compounds prepared by the present invention can significantly reduce the K+ concentration in hepatic stellate cells. The compounds prepared by the present invention can affect the expression of TGF-βR2, p-Smad2, and p-Smad3 in hepatic stellate cells, thereby providing the use of the compounds in drugs that affect the expression of TGF-βR2, p-Smad2, and p-Smad3 in hepatic stellate cells.

[0026] The compound prepared by the present invention can significantly improve the symptoms of rats with liver fibrosis induced by CCl4, thus providing the application of the compound in drugs for improving liver fibrosis induced by CCl4.

[0027] As a further preferred embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0028] As a further preferred embodiment, the pharmaceutically acceptable excipients include any one of a stabilizer, a pH buffer, a surfactant and a pharmaceutically acceptable carrier or a combination thereof.

[0029] As a further preferred embodiment, the pharmaceutical composition may be administered in the form of an injection or oral administration. Injections are preferably dissolved in physiological saline and then administered intravenously. Oral administration includes tablets, capsules, powders, syrups, solutions, aerosols, suspensions, and the like.

[0030] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

[0031] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. For adults, the daily dosage is 25 to 500 μg, preferably 50 to 500 μg, and most preferably 50 to 100 μg. Administration is once daily for three consecutive doses within 72 hours of the pre-myocardial infarction period. The specific dosage should, of course, also be considered in light of factors such as the route of administration and the patient's health status, all of which are within the skill of a skilled physician or pharmacist.

[0032] Compared with the prior art, the present invention is based on the concept of molecular machines and + Given the characteristic that dysfunction of related ion pumps and channels can cause HSC activation, a new class of artificial potassium ion transporters was designed and synthesized based on Centella asiatica, which has anti-inflammatory and antioxidant effects. The synthetic route of the present invention is environmentally friendly, simple and efficient, and the raw materials are readily available, and can be used for the synthesis of similar compounds. Crown ethers are used as potassium ion transport units, and the swinging or relaying of the transport units in the membrane is utilized to achieve transmembrane transport of potassium ions. The present invention uses Centella asiatica, which has multiple pharmacological activities such as anti-inflammatory and antioxidant, as the anchoring unit of the cell membrane, and constructs 10 artificial potassium ion transporters connected to the crown ether through carbon chains of different lengths.

[0033] Further testing of the ion transport activity and selectivity of these compounds revealed that all possessed excellent K+ transport activity, with 6C6 exhibiting high potassium / sodium selectivity. Cell proliferation inhibition experiments revealed that some compounds exhibited significant inhibition of HSC-T6 cell proliferation after activation with TGF-β1. Elisa assays revealed that 6C6 exhibited the best anti-fibrotic marker activity. Subsequent studies of cell morphology and anti-fibrotic mechanisms revealed that these compounds significantly reduced the formation of fibropodia. They also decreased TGF-βR2 expression and inhibited the phosphorylation of p-Smad2 and p-Smad3. In vivo studies significantly reduced CCl4-induced liver fibrosis symptoms. Based on this, artificial ion transporters with both high selectivity and activity are being applied to anti-fibrotic therapies, aiming to provide novel and effective candidate compounds for the treatment of liver fibrosis and to develop reliable anti-fibrotic compounds.

[0034] The present invention overcomes the problems of low activity and insufficient novelty of targets in previous anti-fibrosis drugs, which shows that this type of artificial potassium ion transporter has great application potential in anti-liver fibrosis and is worthy of further development. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the R of the 6C6 cationic pair provided by the present invention M + picture;

[0036] Figure 2 This is a graph showing the effect of 6C6 on membrane integrity provided by the present invention;

[0037] Figure 3 This is a graph showing the inhibition rate of 6C6 provided by the present invention on HSC-T6 cells activated by TGF-β1;

[0038] Figure 4 This is a graph showing the effects of 6C6, 6C8, and 6C10 provided by the present invention on fibrosis markers α-SMA and Col I in HSC-T6 cells stimulated by TGF-β1;

[0039] Figure 5 This is a diagram showing the inhibitory effect of 6C6 provided by the present invention on the cell cycle of HSC-T6 cells activated by TGF-β1;

[0040] Figure 6 This is a graph showing the apoptosis-promoting effect of 6C6 provided by the present invention on HSC-T6 cells activated by TGF-β1;

[0041] Figure 7 This is a diagram showing the effect of 6C6 provided by the present invention on the morphology of HSC-T6 cells after treatment with TGF-β1;

[0042] Figure 8 The 6C6 provided by the present invention is effective for K + Effect diagram of concentration;

[0043] Figure 9 This is a diagram showing the effect of 6C6 on TGFR2, p-Smad2, and p-Smad3 in HSC-T6 provided by the present invention;

[0044] Figure 10 This is a diagram showing Masson and HE staining tests and microscopic examination results of sections of rat liver after 6C6 treatment provided by the present invention. DETAILED DESCRIPTION

[0045] The following examples further describe in detail the artificial potassium ion transporter with anti-hepatic fibrosis activity provided by the present invention, its preparation method, and its application. The following examples are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention.

[0046] A "safe and effective amount" means an amount of active ingredient sufficient to significantly improve the condition without causing serious side effects. Unless otherwise specified, the pharmaceutical compositions provided herein contain 25-500 μg of active ingredient per dose, preferably 50-100 μg per dose. This dosage form is an injection. The drug should be administered at least once per dosing cycle, or once daily for three consecutive days, depending on the area of ​​application.

[0047] "Pharmaceutically acceptable carriers" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available unless otherwise specified.

[0049] Step 1: Preparation of target compound

[0050] The preparation routes of the 10 target compounds are as follows:

[0051]

[0052] (1) Preparation of intermediate XCn-Br:

[0053] Benzoic acid of 15-crown-5 (312 mg, 1 mmol) or 18-crown-6 (356 mg, 1 mmol) was dissolved in 15 mL of acetonitrile. Potassium carbonate (274 mg, 2 mmol) and dibromoalkane (2.13 g, 10 mmol) were added, and the reaction was stirred at 85°C for 12 hours. The reaction mixture was filtered, the solvent was dried by spin drying, dissolved in dichloromethane (150 mL), and washed three times with water (100 mL). The dichloromethane solvent was removed in vacuo to obtain the crude product, which was purified by column chromatography (ethyl acetate) to obtain pure product XCn-Br as a white solid. The specific NMR and mass spectrometric data were consistent with those reported in the literature.

[0054] (2) Preparation of target compound 5C4:

[0055] A suspension of 5C4-Br, Asiatic acid, and potassium carbonate in DMF was heated at 85°C for 12 hours. The solvent was removed in vacuo to afford the crude product, which was dissolved in dichloromethane (50 mL), washed three times with water (50 mL), and purified by column chromatography (methanol:dichloromethane = 1:20) to afford the pure product, 5C4, as a colorless solid. Yield: 336 mg, 51%.

[0056] 1HNMR(600MHz,CDCl3)δ7.68(dd,J=8.4,1.9 Hz,1H),7.53(d,J=2.0 Hz,1H),6.86(d,J=8.5 Hz,1H),5.19(d,J=3.8 Hz,1H),4.38–4.26(m,2H),4.25–4.15(m,4H),4.07–3.98(m,2H),3.92(ddt,J=17.2,10.8,4.4 Hz,4H),3.75(dtt,J=16.0,6.2,3.6 Hz,9H),3.62(td,J=10.5,4.6 Hz,2H),3.31(dd,J=15.2,10.1 Hz,2H),2.22(d,J=11.2 Hz,1H),1.97(dt,J=13.4,6.7 Hz,2H),1.81–1.57(m,11H),1.45(ddd,J=32.1,12.0,4.8 Hz,3H),

[0057] 1.35–1.20(m,6H),1.06–0.98(m,6H),0.94(d,J=6.3Hz,3H),0.85(d,J=6.4Hz,3H),0.79(d,J=11.7Hz,1H),0.75(s,6H),0.65(s,3H). 13 C NMR(150MHz,CDCl3)δ177.74,166.12,152.97,148.2,125.41,124.05,114.29,111.74,79.88,70.65,70.62,70.04,69.99,69.32,69.17,68.72,68.22,68.11,64.35,63.99,52.76,48.34,48.15,47.20,46.20,42.47,42.07,39.51,39.10,38.89,37.81,36.76,32.53,30.66,27.82,26.09,24.96,24.21,23.71,23.30,21.17,18.09,17.09,16.99,12.82.MS-ESI:calculated for[M+Na] + (C 49 H 74 NaO 12 ):m / z 877.5058,found:m / z 877.5056.

[0058] 5C6

[0059] 1 H NMR(600MHz,CDCl3)δ7.66(d,J=8.4Hz,1H),7.53(s,1H),6.85(dd,J=8.5,2.2Hz,1H),5.21(d,J=3.4Hz,1H),4.28(s,2H),4.23–4.16(m,4H),4.05–3.96(m,2H),3.94–3.89(m,4H),3.81–3.71(m,9H),3.58(dd,J=22.1,12.5Hz,2H),3.34(dd,J=13.3,10.2Hz,2H),2.22(d,J=11.2Hz,1H),1.93–1.81(m,4H),1.76(q,J=6.4Hz,3H),1.63(dd,J=19.6,11.1Hz,5H),1.53(t,J=5.4Hz,2H),1.50–1.41(m,6H),1.28(ddt,J=28.3,18.5,10.2Hz,6H),1.05(s,3H),1.03–0.96(m,3H),0.94(d,J=6.1Hz,3H),0.88–0.83(m,6H),0.77(s,3H),0.71(s,3H). 13 C NMR(150MHz,CDCl3)δ177.63,166.33,152.99,148.34,138.38,125.26,123.95,123.17,114.37,111.90,80.52,70.86,70.43,70.20,70.10,69.30,69.17,68.80,68.42,68.33,64.69,64.04,52.80,48.99,48.07,47.33,46.19,42.30,42.12,39.55,39.11,38.87,37.96,36.74,32.66,30.68,28.40,27.88,25.77,25.43,24.22,23.60,23.30,21.19,18.20,17.20,17.02,12.77.MS-ESI:calculated for[M+Na] + (C 51 H 78 NaO 12 ):m / z 905.5391,found:m / z 905.5372.

[0060] 5C8 1 H NMR (600 MHz, CDCl3) δ 7.65 (d, J = 8.7 Hz, 1H), 7.54 (d, J = 9.5Hz, 1H), 5.23 (s, 1H), 4.28 (d, J = 7.4 Hz, 2H), 4.19 (s, 4H),4.00(dt,J=13.7,7.0Hz,2H),3.95–3.90(m,4H),3.75(d,J=7.8Hz,9H),3.59(dd,J=26.9,7.9Hz,2H),3.35(dd,J=24.9,10.3Hz,2H),2.23(d,J=11.7Hz,1H),1.99–1.89(m,4H),1.76(q,J=6.9,6.2Hz,3H),1.70–1.64(m,4H),1.39(dddd,J=53.9,42.2,28.3,17.7Hz,19H),1.07(d,J=9.7Hz,3H),1.05–1.02(m,2H),0.97(d,J=9.0Hz,3H),0.94(d,J=6.2Hz,3H),0.90(s,2H),0.86(s,3H),0.78(d,J=9.6Hz,3H),0.74(d,J=8.8Hz,3H). 13 C NMR(150MHz,CDCl3)δ177.62,166.43,153.05,148.40,138.33,125.26,123.90,123.19,114.50,112.01,80.08,70.96,70.29,70.20,69.89,69.36,69.22,68.89,68.53,64.92,64.19,52.84,48.81,47.40,46.25,42.42,42.14,39.10,38.88,38.03,36.73,32.68,30.69,29.18,29.03,28.76,28.53,27.92,25.99,25.93,24.20,21.19,18.23,17.19,17.12,17.04,12.83.MS-ESI:calculated for[M+H] + (C 53 H 74 NaO 12 ):m / z 911.5885,found:m / z911.5864.

[0061] 5C10

[0062] 1 H NMR(600MHz,CDCl3)δ7.53(s,1H),6.85(d,J=8.5Hz,1H),5.23(d,J=3.7Hz,1H),4.27(t,J=6.7Hz,2H),4.18(q,J=5.0Hz,4H),3.97(q,J=5.9Hz,2H),3.94–3.90(m,4H),3.76(q,J=5.3,3.6Hz,9H),3.57(d,J=10.6Hz,1H),3.38(d,J=9.5Hz,1H),3.35–3.29

[0063] (m, 2H), 2.23 (d, J = 11.2 Hz, 1H), 2.03 – 1.88 (m, 4H), 1.75 (d, J =14.9 Hz, 3H),1.71–1.64(m,2H),1.58(h,J=6.1,5.6Hz,4H),1.53–1.47(m,2H),1.46–1.26(m,19H),1.07(s,3H),1.04(d,J=15.1Hz,2H),0.98(s,3H),0.94(d,J=6.4Hz,3H),0.89(d,J=12.1Hz,2H),0.86(d,J=6.4Hz,3H),0.77(s,3H),0.74(s,3H). 13 C NMR(150MHz,CDCl3)δ177.63,166.49,138.33,153.04,125.26,123.89,123.20,114.54,112.02,79.61,70.96,70.30,70.22,69.36,69.22,69.20,68.90,68.55,68.49,64.98,64.25,52.83,48.52,48.02,47.40,46.29,42.53,42.12,39.57,39.08,38.88,38.03,36.73,32.64,30.69,29.49,29.45,29.30,29.16,28.79,28.56,27.92,26.05,26.03,24.19,23.65,23.34,21.19,18.21,17.16,17.05,12.91.MS-ESI:calculated for[M+H] + (C 55 H 87 O 12):m / z939.6198,found:m / z 939.6183.

[0064] 5C12

[0065] 1 H NMR (600 MHz, CDCl3) δ 7.65 (dd, J = 8.4, 1.9 Hz, 1H), 7.53 (d, J =1.9 Hz, 1H), 6.85 (d, J= 8.4 Hz, 1H), 4.28 (t, J = 6.7 Hz, 2H),4.18(q,J=5.0Hz,4H),3.97(t,J=6.5Hz,2H),3.95–3.89(m,4H),3.75(hept,J=4.3Hz,9H),3.64–3.56(m,2H),3.36(dd,J=16.0,10.0Hz,2H),2.23(d,J=11.3Hz,1H),2.00–1.90(m,4H),1.80–1.73(m,3H),1.69–1.64(m,2H),1.59(dd,J=14.1,7.1Hz,4H),1.52–1.46(m,2H),1.42(t,J=7.6Hz,2H),1.38–1.26(m,21H),1.07(s,3H),1.04(t,J=7.2Hz,2H),1.00(s,3H),0.94(d,J=6.4Hz,3H),0.90(d,J=11.9Hz,2H),0.85(d,J=6.4Hz,3H),0.81(s,3H),0.74(s,3H). 13C NMR(150MHz,CDCl3)δ177.65,166.51,153.05,148.42,125.27,123.90,12.3.23,114.56,112.04,80.14,71.00,70.33,70.23,69.96,69.38,69.24,68.92,68.59,68.51,65.01,64.28,52.82,48.86,48.03,47.43,46.23,42.44,42.14,39.58,39.09,38.88,38.07,36.73,32.69,30.71,29.61,29.57,29.34,29.19,28.80,28.56,27.94,26.06,24.19,23.63,23.35,21.20,18.27,17.19,17.16,17.04,12.83.MS-ESI:calculatedfor[M+Na] + (C 57 H 90 NaO 12 ):m / z 989.6330,found:m / z 989.6307.

[0066] 6C4

[0067] 1H NMR (600 MHz, Chloroform-d) δ 7.47 (d, J = 2.0 Hz, 1H), 6.80 (d, J= 8.5 Hz, 1H), 4.29 (ddd, J = 10.8, 7.9, 5.9 Hz, 1H),4.25–4.19(m,2H),4.13(dd,J=5.2,3.4Hz,2H),4.08(ddd,J=10.3,5.4,2.1Hz,1H),3.99–3.88(m,4H),3.85–3.77(m,3H),3.74–3.69(m,2H),3.70–3.65(m,3H),3.65–3.56(m,8H),3.51(d,J=10.2Hz,2H),1.89(td,J=13.2,4.6Hz,1H),1.75–1.65(m,3H),1.63–1.49(m,6H),1.46–1.38(m,3H),1.38–1.34(m,1H),1.31(d,J=5.2Hz,1H),1.26–1.18(m,4H),1.15–1.06(m,3H),0.94(s,4H),0.87(d,J=6.3Hz,3H),0.77(d,J=6.5Hz,3H),0.73(s,3H),0.58(s,3H),0.52(s,3H). 13 CNMR(151 MHz,Chloroform-d)δ177.84,166.17,152.55,147.94,138.40,125.46,123.88,113.35,111.27,81.17,71.10,70.54,70.41,70.35,70.31,70.25,70.16,69.15,69.06,68.44,68.28,68.07,64.38,64.10,52.71,49.07,48.17,47.21,46.16,42.22,42.04,39.47,39.14,38.92,37.79,36.80,32.55,30.69,27.82,26.29,24.91,24.22,23.70,23.29,21.23,18.11,17.08,17.06,16.90,12.75..MS-ESI:calculated for[M+Na] + (C 51 H 78 NaO 13 ):m / z 921.5340,found:m / z 921.5316.

[0068] 6C6

[0069]

[0070] 1 H NMR (600 MHz, CDCl3) δ 7.67 (dd, J = 8.5, 1.8 Hz, 1H), 7.53 (d, J= 1.8 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 5.19 (t, J= 3.6 Hz, 1H), 4.28 (q, J= 6.6 Hz, 2H), 4.22(dtd,J=12.9,9.0,8.1,3.9Hz,4H),4.07(dt,J=12.2,6.4Hz,2H),3.96(dd,J=8.4,4.7Hz,4H),3.79–3.75(m,4H),3.70(d,J=10.8Hz,8H),3.54(d,J=10.5Hz,1H),3.42(s,1H),3.33(d,J=9.4Hz,1H),3.27(d,J=10.4Hz,1H),2.21(d,J=11.3Hz,2H),2.01–1.94(m,1H),1.87–1.59(m,10H),1.51–1.37(m,7H),1.36–1.25(m,3H),1.25–1.17(m,3H),1.03(s,3H),1.02–0.97(m,2H),0.94(d,J=6.3Hz,4H),0.84(d,J=6.4Hz,3H),0.80(t,J=12.0Hz,1H),0.71(d,J=9.7Hz,5H),0.68(s,3H). 13C NMR(150 MHz,CDCl3)δ177.72,166.32,152.09,147.65,138.49,125.25,123.82,123.20,113.00,111.14,80.47,70.25,70.19,70.14,69.04,68.94,68.30,67.98,64.75,64.08,52.78,48.88,48.10,47.23,45.98,42.29,42.11,39.54,39.14,38.90,37.85,36.77,32.61,30.70,28.69,28.38,27.83,25.84,25.31,24.25,23.63,23.28,21.25,18.14,17.24,17.09,16.92,12.81.MS-ESI:calculated for[M+Na] + (C 53 H 82 NaO 13 ):m / z 949.5653,found:m / z949.5630.

[0071] 6C8

[0072]

[0073] 1H NMR (600 MHz, CDCl3) δ 7.65 (dd, J = 8.4, 1.9 Hz, 1H), 7.53 (d, J =1.9 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 5.22 (t, J = 3.9 Hz, 1H), 4.28 (tt, J= 6.6, 4.2 Hz, 2H),4.21(t,J=4.2Hz,4H),3.99(dt,J=11.6,5.3Hz,2H),3.95–3.90(m,4H),3.78–3.75(m,4H),3.74–3.66(m,9H),3.65–3.57(m,2H),3.35(dd,J=21.5,10.0Hz,2H),2.23(d,J=11.2Hz,1H),1.94(dtt,J=27.6,10.2,5.8Hz,4H),1.76(q,J=6.7Hz,3H),1.69–1.62(m,2H),1.57(dt,J=19.1,5.6Hz,4H),1.49–1.41(m,4H),1.39–1.24(m,13H),1.06(s,3H),1.04–0.97(m,3H),0.95–0.93(m,4H),0.90(d,J=11.7Hz,2H),0.85(d,J=6.2Hz,3H),0.77(s,3H),0.73(s,3H). 13 C NMR(150 MHz,CDCl3)δ177.62,166.43,152.65,148.10,138.33,125.28,123.74,123.12,113.93,111.75,80.49,70.68,70.64,70.57,70.53,70.46,69.32,69.20,68.67,68.52,68.46,64.90,64.17,52.84,49.08,47.41,46.17,42.31,42.15,39.58,39.10,38.88,38.02,36.74,32.70,30.70,29.15,29.00,28.77,28.52,27.90,25.98,25.91,24.21,23.60,23.33,21.19,18.26,17.22,17.08,17.04,12.75.MS-ESI:calculated for[M+Na] + (C 55 H 86 NaO 13):m / z 977.5966,found:m / z977.5945.

[0074] 6C10

[0075]

[0076] 1 H NMR (600 MHz, CDCl3) δ 7.64 (dd, J = 8.4, 2.0 Hz, 1H), 7.53 (d, J =2.0 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 5.23 (d, J = 3.7 Hz, 1H), 4.28 (t, J=6.8Hz,2H),4.21(dt,J=5.4,2.8Hz,4H),3.98(dt,J=6.6,3.3Hz,2H),3.93(dt,J=8.5,4.2Hz,4H),3.78–3.75(m,4H),3.74–3.66(m,9H),3.62(dt,J=13.0,8.3Hz,2H),3.36(dd,J=13.5,10.0Hz,2H),2.23(d,J=11.2Hz,1H),1.94(ddt,J=25.0,9.6,4.3Hz,4H),1.76(qd,J=14.5,13.9,5.6Hz,3H),1.71–1.63(m,3H),1.61–1.55(m,4H),1.45–1.40(m,3H),1.38–1.28(m,15H),1.07(s,3H),1.02(t,J=6.8Hz,2H),0.98(s,3H),0.94(d,J=6.3Hz,3H),0.90(d,J=11.9Hz,1H),0.85(d,J=6.3Hz,3H),0.80(s,3H),0.74(s,3H). 13C NMR(150MHz,CDCl3)δ177.63,166.51,152.74,148.17,125.27,123.75,123.14,114.13,111.85,70.76,70.72,70.63,70.59,69.37,69.26,68.79,68.62,68.52,64.97,64.25,52.83,49.03,48.04,47.44,46.20,42.36,42.14,39.58,39.09,38.88,38.07,36.73,32.70,30.70,29.51,29.45,29.31,29.15,28.81,28.57,27.92,26.06,26.03,24.19,23.62,23.34,21.19,18.29,17.20,17.14,17.04,12.76.MS-ESI:calculated for[M+Na] + (C 57 H 90 NaO 13 ):m / z 1005.6279,found:m / z 1005.6256.

[0077] 6C12

[0078] 1 H NMR (600 MHz, CDCl3) δ7.53(d,J=1.8Hz,1H),6.85(d,J=8.5Hz,1H),5.23(s,1H),4.28(t,J=6.7Hz,2H),4.21(q,J=4.3Hz,4H),3.97(t,J=6.2Hz,2H),3.93(p,J=3.6Hz,4H),3.78–3.73(m,4H),3.70(d,J=17.1Hz,9H),3.58(s,2H),3.35(s,2H),2.29–2.16(m,2H),2.13–1.91(m,11H),1.74(t,J=7.3Hz,2H),1.67(s,2H),1.51–1.39(m,4H),1.36–1.26(m,19H),1.07(s,3H),0.99(s,4H),0.94(d,J=5.7Hz,3H),0.89(d,J=6.6Hz,2H),0.87–0.83(m,3H),0.81(s,3H),0.74(s,3H). 13C NMR(151MHz,Chloroform-d)δ177.72,166.60,152.63,148.08,125.31,123.73,123.07 ,113.76,111.60,80.64,70.71,70.68,70.60,70.57,70.46,69.32,69.20,68.59,68.49 ,65.03,64.31,52.81,48.05,47.43,42.33,42.15,39.58,38.89,38.11,29.69,29.62, 29.41,29.20,28.86,28.58,26.10,23.64,21.26,17.19,17.09,12.77.ESI:calculated for[M+Na] + (C 59 H 94 NaO 13 ):m / z1033.6592,found:m / z 1033.6572.

[0079] Step 2: Transmembrane transport activity assay of compounds

[0080] Ion transport studies based on HPTS vesicle fluorescence experiments

[0081] Egg yolk L-α-phosphatidylcholine (EYPC, 0.6 mL, 25 mg / mL in CHCl₃, Avanti Polar Lipids, USA) was dissolved in CHCl₃ (10 mL). The CHCl₃ was removed under reduced pressure at 35°C. After drying the resulting film under high vacuum at room temperature overnight, the film was hydrated with a HEPES buffer solution (1.5 mL, 10 mM HEPES, 100 mM NaCl, pH = 7.0) containing the pH-sensitive HPTS dye (0.1 mM) and incubated on a shaker at 37°C for 2 hours to produce a milky suspension. The mixture was then subjected to eight freeze-thaw cycles: freezing in liquid nitrogen for 30 seconds followed by heating at 37°C for 1.5 minutes. The vesicle suspension was extruded through a polycarbonate membrane (0.1 μm) to produce a uniform suspension of LUVs with a diameter of approximately 140 nm, encapsulated with HPTS. The suspension of LUVs was dialyzed against the same HEPES buffer solution (300 mL, without HPTS) using membrane tubing (MWCO=10,000) under gentle stirring (300 r / min, 4° C.) for 16 h to remove unencapsulated HPTS to produce LUVs with a lipid concentration of 13 mM.

[0082] A LUV suspension containing HPTS (30 μL, 13 mM in 10 mM HEPES buffer containing 100 mM NaCl, pH = 7.0) was added to a HEPES buffer solution (1.75 mL, 10 mM HEPES, 100 mM NaCl, pH = 8.0) to create a pH gradient for ion transport studies. A solution of the carrier in DMSO was then injected into the suspension under gentle stirring. Immediately after the addition of the carrier, the emission of HPTS was monitored at 510 nm, and excitation at 460 and 403 nm was recorded simultaneously for 300 seconds using a fluorescence spectrophotometer (Hitachi, F-7100 model, Japan). After 300 seconds, an aqueous solution of Triton X-100 (20 μL, 20% v / v) was immediately added to induce the maximum change in the emission of the fluorescent dye. The final transport trajectory was obtained as the ratio value of I460 / I403 and was calculated based on I 460 / I 403 The ratio values ​​were normalized using Equation 1 after adding the sample.

[0083] I f =[(I t -I0) / (I1-I0)] (1)

[0084] 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.

[0085] Table 1. Transmembrane transport activity and selectivity of artificial potassium ion transporters

[0086]

[0087] like Figure 1 As shown, to obtain meaningful R M + To reliably estimate the ion selectivity of 6C6, it is necessary to determine the K + Ions reach saturation within 300 seconds (R K + Following this principle and combining the test results, we tested the transport activity of other alkali metal ions at a concentration of 0.6 μM 6C6. M + value(M + =Li+,Na + , and K + , Cs, etc.), the results are shown in the figure below. It can be found that the transporter showed K + The transmission is highly selective. At this concentration, the Na + / K +The selectivity can reach 25 times.

[0088] 1. Self-quenching CF dye leakage detection

[0089] Egg yolk L-α-phosphatidylcholine (EYPC, 0.6 mL, 25 mg / mL CHCl3, Avanti Polar Lipids, USA) was dissolved in CHCl3 (10 mL). CHCl3 was removed under reduced pressure at 35°C. After drying the resulting film under high vacuum overnight at room temperature, the film was incubated with HEPES buffer solution (1.5 mL, 10 mM HEPES, 100 mM NaCl, pH = 7.5) containing 5(6)-fluorescein (CF, 50 mM) at 37°C for 2 hours in a constant temperature shaker-incubator to obtain a milky suspension. The mixture was then subjected to 8 freeze-thaw cycles: freezing in liquid nitrogen for 30 seconds and then heating at 37°C for 1.5 minutes. The vesicle suspension was extruded through a polycarbonate membrane (0.1 μm) to produce a uniform suspension of large unilamellar vesicles (LUVs) with a diameter of approximately 140 nm, in which the CF dye was encapsulated. The suspension of LUVs was dialyzed against the same HEPES buffer solution (300 mL, without CF dye) using membrane tubing (MWCO=10,000) under gentle stirring (300 r / min, 4°C) for 16 h to remove unencapsulated dye to produce LUVs with a lipid concentration of 13 mM.

[0090] A LUV suspension containing CF (30 μL, 13 mM, prepared with 10 mM HEPES buffer, containing 100 mM NaCl, pH = 7.5) was added to a HEPES buffer solution (1.75 mL, 10 mM HEPES, 100 mM NaCl, pH = 7.5) to create a concentration gradient for CF dye efflux studies. Solutions of potassium ion transporters or melittin in DMSO at different concentrations were then injected into the suspension under gentle stirring. Immediately after the addition of potassium ion transporters or melittin, the emission of CF was monitored at 517 nm using a fluorescence spectrophotometer (Hitachi, F-7100, Japan) and excited at 492 nm for 300 seconds. At t = 300 s, an aqueous solution of Triton X-100 (20 μL, 20% v / v) was immediately added to achieve maximum dye efflux. The final transport trajectory was obtained by normalizing the fluorescence intensity using Equation 2.

[0091] F= [(F t - F0) / (F1- F0)] (2)

[0092] Where F = fractional emission intensity, F t = fluorescence intensity at time t, F1 = fluorescence intensity after adding Triton X-100, F0 = initial fluorescence intensity.

[0093] The fractional change R of each curve was calculated by normalizing the fluorescence intensity before the addition of triton, with the ratio of the reference blank as 0 and the ratio of triton as 1. The Hill coefficient n and EC were given by the Hill equation (3) 50 value.

[0094] Y=1 / (1+ (EC 50 / [C]) n ) (3)

[0095] The experimental results are shown in Figure 2 As shown, the carboxyfluorescein leakage test showed that melittin could form pores >1 nm or effectively disrupt the membrane at low concentrations, increasing solution fluorescence by 13%, 54%, and 86% at 50, 100, and 200 nM, respectively. Compound 6C6 had little effect on solution fluorescence at 2 μM. These results indicate that the presence of 6C6 does not affect the membrane integrity of the vesicles, indicating that 6C6 does not have the activity of directly destroying vesicles.

[0096] 2. Cell Proliferation Inhibition Assay

[0097] The target compound was accurately weighed and diluted to a concentration of 20 μM as a stock solution, filtered and sterilized, and sealed and stored at 4°C for later use. HSC-T6 cells or normal HHL-5 cells were cultured in advance to the logarithmic growth phase, and then the adherent cells in the logarithmic growth phase were digested with trypsin (suspension cells do not need to be digested). 100 μL of cell suspension was added to each well, containing 10 3 -10 4 Cells were seeded in a 96-well plate, and 100 μL of PBS buffer was added to the surrounding wells;

[0098] The cells were cultured in a 37°C cell culture incubator (5% CO2, 90% H2O) until they adhered to the wall; 10 ng / mL TGF-β1 was added for 12 hours, and then the culture medium containing the target compound to be tested was added. The experimental group was added with different concentrations of the test compound (25, 50 μM), 100 μL of the test sample was added to each well, and 4 parallel control wells were set for each concentration; the positive control group was added with the same volume and concentration gradient of asiatic acid solution, and the blank control group was added with 100 μL of culture medium, and the culture was continued for 48 hours; 20 μL of MTT solution was added to each well and the culture was continued for 4 hours; then the culture was terminated and the culture medium in the well was aspirated; 150 μL of DMSO was added to each well and shaken to fully dissolve the crystalline formazan; the absorbance value was measured at 490 nm in a microplate reader, and the inhibition rate of the target compound was calculated according to the formula, and then the software was used to draw some results. Figure 3 It can be seen that 6C10, 6C8, and 6C6 have a significant effect of inhibiting the proliferation of activated HSC-T6 cells, while asiatic acid as a control sample has almost no cell inhibitory activity.

[0099] 3. Detection of fibrosis markers

[0100] The levels of α-SMA and Col I in the samples were determined using commercial kits from Elabscience Biotechnology Co., Ltd. The test steps were as follows: HSC-T6 cells were plated at 2.5×10 4 / mL were seeded in six-well plates and cultured for 24 hours. The cells were divided into high-dose group, low-dose group, negative control group and TGF-β1 group, and the cells were treated with TGF-β1 according to the group and cultured for 24 hours. The cells were then collected from the 6-well plates immediately after lysis with NP40 lysis buffer and centrifuged at 12,000xg for 10 minutes, and the supernatant was aspirated. The total protein content of the samples was measured by BCA protein assay kit. A protein solution with a concentration of 0.1 mg / mL was added to a 96-well plate pre-coated with affinity-purified polyclonal antibodies specific for cytokines, allowed to react for 0.5 hours, and then a final wash was performed to remove any unbound antibody enzyme reagent. After adding the color development solution A, B and the stop solution, the absorbance detected at 450 nm was measured, and the intensity was proportional to the amount of cytokine produced. The cytokine level in each sample was calculated from the standard curve generated by the standard solution. The results are shown in Figure 4 As shown in the figure, it can be seen that compounds 6C10, 6C8, and 6C6 can significantly inhibit the expression of fibrosis markers α-SMA and Col I, and at high concentrations, 6C6 has the best inhibitory effect, which is stronger than asiatic acid. 50(HHL-5) =32.7μM) is safer for normal hepatocytes than 6C8 (IC 50(HHL-5) =19.9 μM) and 6C10 (IC 50(HHL-5) =3.8 μM) is higher, and the SI can reach 2.8 times. In the future, we will focus on studying the anti-fibrotic activity of 6C6.

[0101] 4. Cell Cycle and Apoptosis Assay

[0102] HSC-T6 was seeded in a 6-well plate and cultured for 12 hours. The cells were divided into a high-dose group (20 μM), a low-dose group (10 μM), a negative control group and a TGF-β1 group. After adding TGF-β1 (10 ng / ml) in advance and incubating for 12 hours, compounds of various concentrations were added. The control group cells were added with equal amounts of DMSO and culture medium and cultured for 24 hours. The cells were digested with 0.25% trypsin (0.5 mL), resuspended in culture medium (4 mL), and then centrifuged for 5 minutes (1000 rpm). The cell pellet was washed twice with PBS (2 mL). When performing cell cycle analysis, the cells were fixed in 70% cold ethanol for 4 hours, treated with RNase, and then stained with propidium iodide (PI) for 30 minutes at 37°C in the dark. The DNA content of the cells was analyzed by Beckman flow cytometer. The results are shown in Figure 2. Figure 5 As shown in the results of the present study, the compound can also significantly arrest the cell cycle at the G0 / G1 phase in a concentration-dependent manner.

[0103] For apoptosis analysis, the compounds were added for 48 hours according to the group. The cells were then trypsinized without EDTA, washed three times with PBS, and stained with annexin V-FITC (Yeasen) and PI for 15 minutes in the dark. The percentage of apoptotic cells was determined by flow cytometry within one hour. The results of cell cycle and apoptosis are shown in Figure 2. Figure 6 As shown, the compound can promote the apoptosis of HSC-T6 cells in a concentration-dependent manner. At 20 μM, the apoptosis can reach 35%.

[0104] 5. Cell Morphology Observation Experiment

[0105] HSC-T6 cells in the logarithmic growth phase were cultured at a rate of 2.5×10 4 The cells were seeded at a density of 10 μg / mL in a six-well plate and cultured for 12 hours. The cells were divided into a high-dose group (20 μM), a low-dose group (10 μM), a negative control group, and a TGF-β1 group. After administration according to the group, the cells were cultured for 12 hours, fixed with 4% paraformaldehyde for 10 minutes, washed twice with distilled water, and stained with Giemsa staining solution for 5 minutes. The excess dye was rinsed off with distilled water, and then observed and photographed under an optical microscope. The results are shown in Figure 2. Figure 7 As shown, the addition of TGF-β1 can cause changes in cell morphology due to cell activation, such as increased fiber feet and enlarged cell nuclei, while compound 6G6 can significantly reverse the above changes in a concentration-dependent manner.

[0106] 6. Intracellular K + Concentration level test

[0107] A commercially available EPG probe (Mao Kang Biotechnology) was used to detect intracellular K +Level. HSC-T6 cells were divided into compound group and negative control group. After drug administration according to the group, the cells were plated in 6-well culture plates and incubated for 6 hours. The compound was removed, and the probe was added for staining for 1 hour as instructed. The supernatant was aspirated, washed with PBS, and digested with trypsin. Detection was performed by flow cytometry. Excitation and emission were performed at 485 and 538 nm, respectively, and the data were analyzed using flowJo13.1 software. The experimental results are shown in Figure 2. Figure 8 As shown, the compound can rapidly downregulate intracellular K + levels and is concentration-dependent.

[0108] Western blot analysis of protein expression

[0109] Cells were seeded in a 6-well plate and cultured overnight in a 37°C, 5% CO2 incubator. Compound 6C6 (5, 10, and 20 μM) was then applied for 24 hours. Cells were then washed twice with PBS and lysed on ice for 10 minutes using 300 μL of Solebro High-Performance RIPA Lysis Buffer. Samples were collected and added to SDS-PAGE protein loading buffer (5×). After vortexing and mixing, the samples were denatured in a 95°C water bath for 10 minutes. After cooling, the samples were stored at -20°C for analysis. The gel plate was sealed with plastic wrap. SDS-PAGE separating and stacking gels were prepared based on the molecular weight of the protein to be analyzed. A comb was then inserted, placed vertically upward, and allowed to stand for several minutes. After sufficient solidification, the plastic wrap and comb were removed. The prepared gel plate was inserted into the electrophoresis tank, and equal volumes of sample and marker were added to each well. Electrophoresis was performed under gradient electrophoresis conditions. After the electrophoresis is completed, the gel is peeled off, and the 0.22μm PVDF membrane is activated in methanol for 5 minutes. The separated protein samples are transferred to the activated PVDF membrane using a wet transfer electrophoresis tank. After the transfer is completed, the PVDF membrane is placed in 5% skimmed milk powder in TBST blocking solution and blocked at room temperature for 1.5 hours. Wash the membrane 3 times with TBST buffer, each for 10 minutes. Place the PVDF membrane in the corresponding primary antibody diluted in an appropriate proportion and incubate at 4°C overnight. Wash the membrane 3 times with TBST buffer, each for 10 minutes. Add HRP-labeled IgG secondary antibody diluted in an appropriate proportion and incubate on a shaker at room temperature for 1.5 hours. After the antibody incubation is completed, wash the membrane again with TBST buffer 3 times, each for 10 minutes. Add ECL chemiluminescent solution and use the chemiluminescent module of Tianneng multifunctional imager for imaging. The results are as follows Figure 9 As shown, the selected compound 6C6 inhibited the expression of TGF-βR2 and downstream p-Smad 2 and p-Smad 3 proteins in a concentration-dependent manner.

[0110] In vivo anti-fibrotic activity of compounds

[0111] All animal procedures were approved by the Institutional Ethics Committee of Xiamen University. Male Wistar rats (180 ± 10 g) were randomly divided into four groups: (a) a normal control group (0.5 mL / kg bw) intraperitoneally injected with olive oil twice weekly for four weeks; (b) a CCl4 group; (c) a low-dose group (5 mg / kg); and (d) a high-dose group (15 mg / kg). All groups, except the normal control group, received CCl4 (2 mL / kg bw, 40% CCl4 / vegetable oil) intraperitoneally twice weekly for four consecutive weeks. After establishing a rat liver fibrosis model, the rats were intraperitoneally injected with the compound according to grouping. The rats were intraperitoneally administered with the compound once daily for two weeks. At the end of the two weeks, each rat was euthanized and its organs and tissues were removed. The livers of the CCl4 group were significantly paler, more enlarged, with a rougher surface and a brittle texture compared to the normal group. However, after 6C6 treatment, the livers of the rats became darker, smoother, and more firm, and this improvement was concentration-dependent. Liver samples were collected and sent to Sevier Biotech for Masson and HE staining tests. The results of microscopic examination of the sections were as follows: Figure 10 shown.

[0112] HE staining and collagen fiber staining showed that the liver lobule structure of the normal group was intact, and the liver cells were neatly arranged around the central vein. The liver cells were normal, without fatty degeneration, and arranged in a cord-like manner. The cells in the CCl4 model group were disorderly arranged, the tissue was obviously damaged, and the intracellular fatty degeneration was obvious and necrotic. At the same time, the connective tissue in the liver was obvious and necrotic. At the same time, the connective tissue in the liver proliferated and aggregated, fibrous septa were formed, and inflammatory cell infiltration occurred. In Masson staining, the collagen deposition in the liver of the liver fibrosis group was significantly increased compared with the normal liver. The compound can effectively inhibit the damage caused by CCl4 at low doses.

[0113] Finally, it is necessary to explain here that the above embodiments are only used to further illustrate the technical solution of the present invention in detail and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above content of the present invention all fall within the scope of protection of the present invention.

Claims

1. An artificial potassium ion transporter with anti-hepatic fibrosis activity, characterized in that: The general structural formula of the compound of the artificial potassium ion transporter with anti-hepatic fibrosis activity is as follows: Wherein, m is 2, and n is 2 to 4. The compound uses crown ether as the potassium ion transport unit and asiatic acid as the cell membrane anchoring unit. Three artificial potassium ion transporters are constructed by connecting the crown ether with carbon chains of different lengths, specifically: 6C6(4), 6C8(6) and 6C10(8). The structural formulas are as follows: or their stereoisomers, tautomers, and pharmaceutically acceptable salts.

2. The artificial potassium ion transporter with anti-hepatic fibrosis activity according to claim 1, characterized in that The synthetic route of the compound having anti-hepatic fibrosis activity artificial potassium ion transporter is as follows: The synthesis process is divided into two steps. In the first step, dibromoalkanes of different lengths are reacted with crown benzoic acid to obtain intermediate compounds XCn-Br, where n = 2 to 4 and X = 6. In the second step, XCn-Br in the first step is reacted with asiatic acid through an alkylation reaction to obtain the target compounds 6C6, 6C8 and 6C10.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the artificial potassium ion transporter with anti-liver fibrosis activity according to claim 1 as an active ingredient, and further comprises pharmaceutically acceptable excipients.

4. The pharmaceutical composition according to claim 3, characterized in that The compound is used in preparing drugs for treating and / or preventing liver fibrosis.

5. The pharmaceutical composition according to claim 3, characterized in that The compound is used in drugs for inhibiting the proliferation and differentiation of hepatic stellate cells HSC-T6 activated by TGF-β1.

6. The pharmaceutical composition according to claim 3, characterized in that Application of the compound in drugs for reducing the expression of fibrosis markers α-SMA and Col I.

7. Use of the artificial potassium ion transporter with anti-hepatic fibrosis activity according to any one of claims 1 to 2 or the pharmaceutical composition according to any one of claims 4 to 6 in the preparation of a drug for increasing K + activity, and / or for inhibiting hepatocellular fibrosis.

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