A glutathione dimer with active oxygen response and preparation and application thereof

Abstract

The application discloses glutathione dimers with active oxygen response and preparation and application thereof, wherein the glutathione dimers are formed by connecting two glutathione (GSH) monomers through ketone thiol acetal (TK) containing active oxygen response; the glutathione dimers can quickly respond to and remove excessive active oxygen (ROS) at an inflammation site, release GSH, regulate the ROS level in an inflammation microenvironment, play a therapeutic role while releasing drugs, synergistically enhance the antioxidant therapeutic effect, and have important application potential in antioxidant and inflammatory disease treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to a glutathione dimer, in particular to a glutathione dimer with active oxygen response, and also relates to the preparation and application of the above-mentioned glutathione dimer. BACKGROUND

[0002] Studies have shown that inflammation-related diseases are often accompanied by oxidative stress phenomena, a significant feature of which is an increase in the level of reactive oxygen species (ROS). These ROS species, including superoxide anions, hydrogen peroxide and hydroxyl radicals, are byproducts of normal metabolic processes in living organisms. Under normal physiological conditions, ROS plays an important role in cell signaling, gene expression regulation and host defense mechanisms. However, once the balance between ROS generation and clearance is disrupted, excessive accumulation of ROS occurs, triggering oxidative stress, which causes damage to cell structures, proteins, lipids and nucleic acids.

[0003] Oxidative stress imbalance is closely related to the development of various pathological conditions. For example, in liver diseases, excessive accumulation of ROS attacks the lipids, proteins and DNA of cell membranes, leading to cell damage, and thus to diseases such as hepatitis, liver fibrosis and liver cancer. In cardiovascular diseases, elevated levels of ROS are associated with pathologies such as atherosclerosis, hypertension and myocardial infarction. In addition, ROS is considered a key factor in driving inflammatory diseases such as rheumatoid arthritis and asthma. Glutathione (GSH) is an endogenous antioxidant tripeptide that also participates in redox balance and cell signal regulation. It protects cells from oxidative damage by reducing H2O2 and eliminating ROS and nitrogen radicals. In clinical use, oral glutathione supplements are one of the most common forms, usually in the form of capsules, tablets or powders. Glutathione is also used to enhance immune function and as an adjunct therapy for liver diseases, particularly in the treatment of chronic hepatitis B, cirrhosis and other liver diseases. For example, R. Schauer (World J. Gastroenterol., 2004, 10(6), 864-70.) et al. treated rats with 100 mg / kg after liver transplantation reperfusion injury, and the ALT levels were significantly lower than the control group. Electron microscopy morphological results confirmed that GSH treatment prevented the separation of sinusoidal endothelial cells (SEC) and the loss of hepatocyte microvilli and mitochondrial swelling.

[0004] TK (thio-ketone) is a sub-class of sulfur-containing groups, which is cleaved into thiol-containing groups and acetone when exposed to ROS (reactive oxygen species). In the current study, TK is widely used to develop ROS-responsive drug delivery systems due to its biodegradability and non-toxicity. For example, TK-containing nanocarriers (such as polymer nanoparticles) and TK-modified liposomes. These drug preparations are stable under normal physiological conditions, and the ROS in the microenvironment of diseases such as tumors can degrade the carrier to effectively release the drug. However, the existing drug delivery system is complex and difficult to achieve. In addition, TK is also used as an antioxidant because it reacts with ROS and removes ROS. For example, Shammy et al. (Advanced Therapeutics, 2023, 6(1)) used TK as an antioxidant to reduce ROS-mediated oxidative damage to islets and significantly protect the viability and function of islet cells. This treatment method improves the effect of marginal islet transplantation, and the islet recipients treated with TK reach normal blood glucose levels within 2 days after transplantation, while the islet recipients without TK treatment fail to reach normal blood glucose levels. Compared with the control group, TK-treated islets show a significantly higher rate of diabetes reversal. In the above examples, TK is only introduced as a responsive linker group into the carrier system for controlled drug delivery or directly used as an antioxidant, and its application is still relatively narrow.

[0005] Compounds with similar ROS responsiveness include thioether dioxide (TED), selenoether (Se-Ether) groups, and thioether-ether groups. However, the metabolic products of the above-mentioned compounds, thioether dioxide, may have potential toxicity in vivo; the use of selenoether groups is limited due to the potential toxicity of selenide at high concentrations; the biocompatibility and stability of the cleavage products of thioether-ether groups under ROS conditions are relatively insufficient; and active oxygen-responsive compounds suitable for practical use still need to be studied. SUMMARY

[0006] The purpose of the present application is to provide an active oxygen-responsive glutathione dimer, and also to provide a preparation method and application of the above-mentioned glutathione dimer.

[0007] Technical solution: The active oxygen-responsive glutathione dimer according to the present application is shown as formula I: .

[0008] The preparation method of the above-mentioned glutathione dimer comprises the following steps: freeze-drying glutathione, mixing with an organic solvent, then adding trifluoroacetic acid, stirring at room temperature overnight, precipitating with cold ether to obtain white precipitate, filtering and drying to obtain the active oxygen-responsive glutathione dimer.

[0009] The preparation method of the glutathione dimer, or the following steps are adopted: glutathione is freeze-dried, mixed with an organic solvent, stirred, heated, and magnesium bromide ether solution is added to obtain a mixture; 2,2-dimethoxypropane dissolved in anhydrous acetonitrile is added dropwise into the mixture, stirred overnight, concentrated by rotary evaporation, precipitated with cold ether, and vacuum dried to obtain the glutathione dimer with active oxygen response.

[0010] The glutathione is reduced glutathione powder, the molecular weight is 307.32 Da; the molecular weight of acetone is 58.08 Da, and the structure is as shown in formula I. .

[0011] The organic solvent is acetone or anhydrous acetonitrile, and the molar ratio of glutathione to the organic solvent is 1:1-5; and further preferably 1:2.

[0012] The preparation method of the glutathione dimer, or the following steps are adopted: glutathione is freeze-dried, mixed with an organic solvent, stirred, heated, and magnesium bromide ether solution is added to obtain a mixture; 2,2-dimethoxypropane dissolved in anhydrous acetonitrile is added dropwise into the mixture, stirred overnight, concentrated by rotary evaporation, precipitated with cold ether, and vacuum dried to obtain the glutathione dimer with active oxygen response.

[0013] The glutathione is oxidized glutathione, and the structure is as shown in formula III. .

[0014] The molar ratio of the glutathione to acetone is 1:1-5.

[0015] The glutathione dimer can also be applied in the preparation of a medicine for inhibiting oxidative stress and / or treating inflammation.

[0016] The glutathione dimer is broken by responding to ROS at an inflammation site, and endogenous antioxidant tripeptide GSH is released to achieve the effect of antioxidant and / or inflammation treatment.

[0017] The principle of the application is that two glutathione monomers are connected through a ketone thioacetal bond containing active oxygen response to form a ROS-responsive dimer, and the two functions of TK are combined with GSH to obtain a ROS-responsive GSH dimer prodrug. On the one hand, the dimer responds to ROS at an inflammation site to break the ketone thioacetal bond and remove ROS at the site; on the other hand, the dimer can release endogenous antioxidant tripeptide GSH to further remove active oxygen species. Therefore, the two functions of TK are combined with GSH to obtain a ROS-responsive GSH dimer prodrug, and the glutathione dimer with active oxygen response has important application potential as an antioxidant in liver diseases and various inflammatory diseases.

[0018] Beneficial effects: Compared with the prior art, the glutathione dimer of the present application has the following remarkable advantages: the ketone thioacetal bond of the glutathione dimer of the present application can be broken by responding to ROS at the inflammation site, ROS can be removed, and endogenous antioxidant tripeptide GSH can be released to further remove active oxygen species, which significantly improves its application in inhibiting oxidative stress and treating inflammation, makes it quickly release drugs at sites containing ROS, thereby producing a high-efficiency therapeutic effect, and has great application prospects in the fields of intelligent responsiveness and controlled release of drugs. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The hydrogen nuclear magnetic spectrum of the glutathione dimer with active oxygen response of the present application;

[0020] Figure 2 The hydrogen nuclear magnetic spectrum of the glutathione dimer with active oxygen response of the present application in an active oxygen environment;

[0021] Figure 3 The concentration-DPPH free radical scavenging rate result of the glutathione dimer with active oxygen response of the present application;

[0022] Figure 4 The time-DPPH free radical scavenging rate result of the glutathione dimer with active oxygen response of the present application. DETAILED DESCRIPTION

[0023] The technical solutions of the present application will be further described below in combination with examples. The test materials used in the examples can be purchased through conventional channels.

[0024] Example 1

[0025] The preparation method of the glutathione dimer with active oxygen response of the present application comprises the following steps: ;

[0026] Take reduced glutathione (GSH, 900 mg, 2.93 mmol) and dissolve it in 1M hydrochloric acid, freeze-dry, then mix with acetone (1.2 mL, 16 mmol), and then add a catalytic amount of trifluoroacetic acid (5 μL), stir at room temperature overnight, precipitate with cold ether, obtain white precipitate, filter, dry, and obtain a white powder with a yield of 76%.

[0027] Example 2

[0028] The preparation method of the glutathione dimer with active oxygen response of the present application comprises the following steps:

[0029] Anhydrous acetonitrile (ACN) and freeze-dried reduced glutathione (GSH, 900 mg, 2.93 mmol) were continuously stirred in a reaction bottle, heated to 80°C, 5 μL of magnesium bromide ether solution (MgBr2 / Et2O) was added, 2,2-dimethoxypropane (312.45 mg, 3 mmol) dissolved in anhydrous acetonitrile was added dropwise into the mixture, and stirred overnight. Concentrated by rotary evaporation, precipitated with cold ether, and dried in vacuum. A white powder with a yield of 63% was obtained.

[0030] Example 3

[0031] The preparation method of the glutathione dimer with active oxygen response of the present application comprises the following steps: ;

[0032] Take oxidized glutathione (GSSG, 1.3 g, 2.12 mmol) and acetone (1.2 mL, 16 mmol), add tributylphosphine (6 μL) dropwise, stir at room temperature overnight, separate the GSH dimer by column chromatography, collect the product, concentrate under reduced pressure, and dry in vacuum to obtain a white powder.

[0033] Take the white powder prepared in Examples 1-3 for nuclear magnetic resonance characterization, and the results are shown in Figure 1 It can be seen that the glutathione dimer with active oxygen response of the present application is prepared.

[0034] The glutathione dimer of the present application is subjected to active oxygen response research:

[0035] Take the prepared glutathione dimer, add 1M H2O2 and incubate for 12 h, freeze and vacuum dry, and compare the hydrogen nuclear magnetic spectrum with that of reduced glutathione.

[0036] The hydrogen nuclear magnetic spectrum of the glutathione dimer and the glutathione dimer in an environment of excessive high-concentration H2O2 is shown in Figure 2 The absorption peak of hydrogen on the TK methyl group at δ1.3 is significantly weakened in the nuclear magnetic spectrum after the addition of hydrogen peroxide, indicating the breakage of TK, and the peak around δ2.1 proves the generation of acetone.

[0037] The glutathione dimer of the present application is subjected to DPPH (2-2 diphenyl hydrazy) scavenging capacity determination:

[0038] A solution of DPPH with a concentration of 0.2 mmol / L (mM) was prepared using anhydrous ethanol. 2 mL of glutathione dimer solutions with different concentrations (0, 80, 160, 320, 640, 1000, 2000 μM) were measured, 2 mL of DPPH solution was added, and the reaction was carried out at room temperature for 30 min in the dark. Anhydrous ethanol was used as a blank control, and the absorbance value was measured at 517 nm. The same concentration of glutathione solution was used as a positive control. Each sample was measured in triplicate, and the DPPH solution clearance rate was calculated according to the following formula: clearance rate (%) = [1-(Ai-Aj / A0)]*100%, where Ai represents the absorbance value of the test solution, Aj represents the background value of the test solution, and A0 represents the control value without the test solution.

[0039] A solution of DPPH with a concentration of 0.2 mmol / L (mM) was prepared using anhydrous ethanol. 2 mL of glutathione dimer solutions with different concentrations (0, 80, 160, 320, 640, 1000, 2000 μM) were measured, 2 mL of DPPH solution was added, and the reaction was carried out at room temperature for 30 min in the dark. Anhydrous ethanol was used as a blank control, and the absorbance value was measured at 517 nm. The same concentration of glutathione solution was used as a positive control. Each sample was measured in triplicate, and the DPPH solution clearance rate was calculated according to the following formula: clearance rate (%) = [1-(Ai-Aj / A0)]*100%, where Ai represents the absorbance value of the test solution, Aj represents the background value of the test solution, and A0 represents the control value without the test solution.

[0040] The results of the DPPH clearance ability of GSH dimer are shown in Figure 3 : Within the concentration range of 0 to 2000 µM, both GSH and GSH dimer can clear DPPH free radicals. When the concentration is 600 µM and the incubation time is 60 minutes, the DPPH clearance rate of GSH dimer is 21.1% higher than that of GSH, and 5.9% higher than that of GSH with a concentration of 1200 µM Figure 4 , and the free radical clearance effect of GSH dimer is better than that of 2 times GSH. The dimer can break the thio-ketone bond (TK) in response to excess ROS at the inflammation site, thereby clearing part of the ROS. At the same time, this process releases monomeric glutathione, which further clears excess ROS at the inflammation site. The dimer can effectively exert the synergistic effect of TK and glutathione monomer, and can significantly enhance the antioxidant treatment effect.

[0041] Therefore, GSH dimer can clear DPPH free radicals through thio-ketone reaction, and the released reducing GSH can also effectively clear DPPH free radicals.

Claims

1. A glutathione dimer having a reactive oxygen response, characterized in that, The dimer is shown as formula I: 。 2. A method of producing the glutathione dimer of claim 1, characterized by, The method comprises the following steps: The reduced glutathione is freeze-dried, mixed with acetone, and then trifluoroacetic acid is added. The mixture is stirred at room temperature overnight, precipitated with cold ether, and then filtered and dried to obtain the active oxygen-responsive glutathione dimer.

3. A method for preparing the glutathione dimer according to claim 1, characterized in that, The method comprises the following steps: The reduced glutathione is freeze-dried, mixed with anhydrous acetonitrile, and then heated and stirred. Magnesium bromide ether solution is added to obtain a mixture. 2,2-dimethoxypropane dissolved in anhydrous acetonitrile is added dropwise into the mixture, which is stirred overnight. The mixture is concentrated by rotary evaporation, precipitated with cold ether, and then dried in vacuum to obtain the active oxygen-responsive glutathione dimer.

4. A method of producing the glutathione dimer of claim 1, wherein, The method comprises the following steps: The oxidized glutathione is mixed with acetone, and then tributylphosphine is added dropwise. The mixture is stirred at room temperature overnight. The product is separated and collected by column chromatography, concentrated under reduced pressure, and then dried in vacuum to obtain the active oxygen-responsive glutathione dimer.

5. Use of the glutathione dimer of claim 1 in the preparation of a drug for resisting oxidation and / or treating inflammation.

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