Glutathione dimer with active oxygen response as well as preparation and application of glutathione dimer

Abstract

The invention discloses a glutathione dimer with active oxygen response as well as preparation and application of the glutathione dimer. The glutathione dimer is formed by connecting two glutathione (GSH) monomers through a ketal thiol bond (TK) with active oxygen response. The glutathione dimer disclosed by the invention can quickly respond to and remove excessive reactive oxygen species (ROS) at an inflammation part, release GSH and regulate the ROS level in an inflammation microenvironment, plays a treatment role while releasing a medicine, synergistically enhances an anti-oxidation treatment effect and has important application potential in anti-oxidation and treatment of inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to a glutathione dimer, in particular to a glutathione dimer with active oxygen response, and also to the preparation and application of the glutathione dimer. Background Art

[0002] Studies have shown that inflammatory diseases are often accompanied by oxidative stress, a notable feature of which is an increase in the level of reactive oxygen species (ROS). These ROS species, including free radicals such as superoxide anions, hydrogen peroxide, and hydroxyl radicals, are byproducts of normal metabolic processes in organisms. Under normal physiological conditions, ROS play an important role in cell signaling, gene expression regulation, and host defense mechanisms. However, once the balance between ROS generation and clearance mechanisms is disrupted, it will lead to excessive accumulation of ROS, triggering oxidative stress, thereby damaging cell structures, proteins, lipids, and nucleic acids.

[0003] Imbalanced oxidative stress is closely linked to the development of various pathological conditions. For example, in liver disease, excessive accumulation of ROS attacks cell membrane lipids, proteins, and DNA, leading to cell damage and, in turn, diseases such as hepatitis, liver fibrosis, and liver cancer. In cardiovascular disease, elevated ROS levels are associated with pathologies such as atherosclerosis, hypertension, and myocardial infarction. Furthermore, ROS are 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 homeostasis and cell signaling regulation. It protects cells from oxidative damage by reducing H₂O₂ and scavenging ROS and nitrogen free radicals. In clinical use, oral glutathione supplementation is one of the most common forms, typically in capsules, tablets, or powder. Glutathione is also used to enhance immune function and as an adjunct therapy for liver disease, particularly in the treatment of chronic hepatitis B, cirrhosis, and other liver diseases. For example, R. Schauer et al. (World J. Gastroenterol., 2004, 10(6), 864-70.) found that the ALT levels of rats with post-liver transplantation reperfusion injury treated with 100 mg / kg GSH were significantly lower than those of the control group. Electron microscopic morphological results confirmed that GSH treatment prevented the separation of hepatic sinusoidal endothelial cells (SEC) and the loss of hepatocyte microvilli and mitochondrial swelling.

[0004] The TK (thioketal) group is a subclass of sulfur-containing groups that is cleaved into thiol groups and acetone when exposed to ROS (reactive oxygen species). In current research, TK is widely used to develop ROS-responsive drug delivery systems due to its biodegradability and non-toxicity. For example, nanocarriers containing TK groups (such as polymer nanoparticles) and TK-modified liposomes. These drug preparations are stable under normal physiological conditions, but when the carriers are degraded under the action of ROS in disease microenvironments such as tumors, the drugs can be effectively released. However, existing drug delivery systems are relatively complex and difficult to implement. In addition, TK is also used as an antioxidant because it reacts with ROS and then removes ROS. For example, Shammy et al. (Advanced Therapeutics, 2023, 6(1)) used TK as an antioxidant to reduce ROS-mediated oxidative damage to pancreatic islets and significantly protected the viability and function of pancreatic islet cells. This treatment improved the efficacy of marginal islet transplantation, with TK-treated mouse islet recipients achieving normoglycemia within 2 days of transplantation, whereas non-TK-treated islet recipients failed to achieve normoglycemia. Compared with the control group, TK-treated islets showed a significantly improved diabetes reversal rate. In these examples, TK was only introduced as a responsive linker into carrier systems for controlled drug delivery or used directly as an antioxidant, but its applications remain relatively narrow.

[0005] Compounds with similar ROS-responsiveness include thioether dioxide (TED), selenoether (Se-Ether) groups, and thioether-Ether (Thioether-Ether) groups. However, the metabolites of thioether dioxides may be potentially toxic in vivo; the use of selenoether groups is limited because selenides may be toxic to cells at high concentrations; and the cleavage products of thioether groups under ROS conditions are relatively incompatible with biocompatibility and stability. Compounds suitable for practical ROS-responsiveness remain under investigation. Summary of the Invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a glutathione dimer with active oxygen response, and also provide a preparation method and application of the above glutathione dimer.

[0007] Technical solution: The glutathione dimer with active oxygen response of the present invention is shown in Formula I:

[0008]

[0009] The method for preparing the glutathione dimer comprises the following steps: freeze-drying glutathione, mixing it with an organic solvent, adding trifluoroacetic acid, stirring it at room temperature overnight, precipitating it with cold ether to obtain a white precipitate, filtering it, and drying it to obtain a glutathione dimer that responds to active oxygen.

[0010] The preparation method of the above-mentioned glutathione dimer can alternatively adopt the following steps: freeze-dried glutathione, mixed with an organic solvent, stirred, heated, and added with an ethereal solution of magnesium bromide to obtain a mixture; 2,2-dimethoxypropane dissolved in anhydrous acetonitrile is added dropwise to the mixture, stirred overnight, concentrated by rotary evaporation, precipitated with cold ether, and vacuum dried to obtain a glutathione dimer with active oxygen response.

[0011] The glutathione is reduced glutathione powder with a molecular weight of 307.32 Da; the molecular weight of acetone is 58.08 Da, and the structure is shown in Formula I:

[0012]

[0013] Wherein, the organic solvent is acetone or anhydrous acetonitrile, and the molar ratio of glutathione to the organic solvent is 1:1 to 5, and more preferably 1:2.

[0014] The above-mentioned method for preparing the glutathione dimer can alternatively be performed by the following steps: glutathione and acetone are mixed, tributylphosphine is added dropwise, and the mixture is stirred and reacted at room temperature overnight. The product is separated and collected by column chromatography, concentrated under reduced pressure, and dried in vacuo to obtain the glutathione dimer having active oxygen response.

[0015] Wherein, the glutathione is oxidized glutathione, and its structure is shown in Formula III:

[0016]

[0017] Wherein, the molar ratio of glutathione to acetone is 1:1-5.

[0018] The glutathione dimer can also be used in the preparation of drugs for inhibiting oxidative stress and / or treating inflammation.

[0019] The glutathione dimer breaks the thioketal bond in response to ROS at the inflammatory site and releases endogenous antioxidant tripeptide GSH to achieve the effect of antioxidant and / or inflammatory treatment.

[0020] Principle of the Invention: This invention connects two glutathione monomers via a thiol-ketal bond that is responsive to reactive oxygen species (ROS), forming a ROS-responsive dimer. This is the first time that the two functions of TK have been combined, resulting in a covalent bond with GSH to create a ROS-responsive GSH dimer prodrug. On the one hand, the dimer responds to ROS at the site of inflammation by cleaving the thiol-ketal bond and scavenging ROS at that site; on the other hand, the dimer releases the endogenous antioxidant tripeptide GSH, further scavenging reactive oxygen species. Therefore, this invention represents the first time that the two functions of TK have been combined, resulting in a covalent bond with GSH to create a ROS-responsive GSH dimer prodrug. The present ROS-responsive glutathione dimer has significant potential as an antioxidant in the treatment of liver disease and various inflammatory diseases.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the glutathione dimer of the present invention can break the ketal thiol bond by responding to ROS at the inflammatory site, thereby clearing ROS and releasing endogenous antioxidant tripeptide GSH to further clear reactive oxygen species, significantly improving its application in inhibiting oxidative stress and treating inflammation, enabling it to quickly release drugs at sites containing ROS, thereby producing an efficient therapeutic effect, and has great application prospects in the fields of intelligent responsiveness and controlled drug release. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a hydrogen nuclear magnetic spectrum of the glutathione dimer with active oxygen response of the present invention;

[0023] Figure 2 The 1H NMR spectrum of the glutathione dimer with active oxygen response under active oxygen environment of the present invention;

[0024] Figure 3 The results of the present invention are the concentration of glutathione dimer with active oxygen response and DPPH free radical scavenging rate;

[0025] Figure 4 This is the result of the glutathione dimer time-DPPH free radical scavenging rate with active oxygen response of the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below with reference to the examples. The test materials used in the examples can all be purchased through conventional channels.

[0027] Example 1

[0028] The method for preparing the active oxygen-responsive glutathione dimer of the present invention comprises the following steps:

[0029]

[0030] Reduced glutathione (GSH, 900 mg, 2.93 mmol) was dissolved in 1 M hydrochloric acid, lyophilized, mixed with acetone (1.2 mL, 16 mmol), and then a catalytic amount of trifluoroacetic acid (5 μL) was added. The mixture was stirred at room temperature overnight and precipitated with cold ether to obtain a white precipitate. The precipitate was filtered and dried to obtain a white powder with a yield of 76%.

[0031] Example 2

[0032] The method for preparing the active oxygen-responsive glutathione dimer of the present invention comprises the following steps:

[0033]

[0034] Anhydrous acetonitrile (ACN) and lyophilized reduced glutathione (GSH, 900 mg, 2.93 mmol) were stirred continuously in a reaction flask, heated to 80°C, and 5 μL of magnesium bromide in ether (MgBr2 / Et2O) was added. 2,2-dimethoxypropane (312.45 mg, 3 mmol) dissolved in anhydrous acetonitrile was added dropwise to the mixture, and stirred overnight. The product was concentrated by rotary evaporation, precipitated with cold ether, and dried under vacuum. A white powder with a yield of 63% was obtained.

[0035] Example 3

[0036] The method for preparing the active oxygen-responsive glutathione dimer of the present invention comprises the following steps:

[0037]

[0038] Oxidized glutathione (GSSG, 1.3 g, 2.12 mmol) and acetone (1.2 mL, 16 mmol) were mixed, tributylphosphine (6 μL) was added dropwise, and the mixture was stirred and reacted at room temperature overnight. GSH dimers were separated by column chromatography, and the collected product was concentrated under reduced pressure and dried in vacuo to obtain a white powder.

[0039] The white powders obtained in Examples 1 to 3 were subjected to NMR characterization. Figure 1 As shown, it can be seen that the glutathione dimer responsive to active oxygen of the present invention was prepared.

[0040] The glutathione dimer of the present invention was studied for its responsiveness to active oxygen species:

[0041] The prepared glutathione dimer was taken, 1 M H2O2 was added and incubated for 12 h, and then freeze-dried in a vacuum, and the H NMR spectrum was compared with that of reduced glutathione.

[0042] The hydrogen nuclear magnetic spectrum of glutathione dimer and glutathione dimer in an excess high concentration H2O2 environment is as follows Figure 2As shown, the absorption peak at δ1.3 of the glutathione dimer is the characteristic absorption peak of hydrogen on the TK methyl group. In the NMR spectrum after the addition of hydrogen peroxide, the absorption peak at δ1.3 is significantly weakened, indicating the breakage of TK, and the peak appearing at around δ2.1 proves the formation of acetone.

[0043] The glutathione dimer of the present invention was tested for its DPPH (2-2diphenyl hydrazy) scavenging ability:

[0044] Prepare a 0.2 mmol / L (mM) DPPH solution in anhydrous ethanol. Measure 2 mL of glutathione dimer solution at various concentrations (0, 80, 160, 320, 640, 1000, and 2000 μM) and add 2 mL of DPPH solution. Incubate in the dark for 30 minutes at room temperature. Measure the absorbance at 517 nm using anhydrous ethanol as a blank control. A glutathione solution of the same concentration serves as a positive control. Repeat the measurement three times for each sample. Calculate the DPPH clearance rate using the following formula: Clearance (%) = [1-(Ai - Aj / A0)] * 100%, where Ai represents the absorbance after adding the test solution; Aj represents the background value of the test solution; and A0 represents the control value without the test solution.

[0045] Prepare a 0.2 mmol / L (mM) DPPH solution with anhydrous ethanol, measure 2 mL of 600 μM glutathione dimer solution and 600 μM and 1200 μM glutathione solutions, add 2 mL of DPPH solution, and measure the absorbance at 517 nm at 10, 20, 30, 40, 50, and 60 min using anhydrous ethanol as a blank control according to the above requirements.

[0046] The results of GSH dimer scavenging ability for DPPH are as follows Figure 3 As shown in the figure: In the concentration range of 0 to 2000 μM, both GSH and GSH dimer can scavenge DPPH free radicals. When the concentration is 600 μM and the incubation time is 60 minutes, the DPPH scavenging rate of GSH dimer is 21.1% higher than that of GSH, and 5.9% higher than that of GSH at a concentration of 1200 μM. Figure 4 ), the free radical scavenging effect of GSH dimer is superior to that of 2-fold GSH. This dimer responds to excess ROS at the site of inflammation by cleaving the thiol ketal (TK) bond within it, thereby scavenging some ROS. Simultaneously, this process releases monomeric glutathione, further scavenging excess ROS at the site of inflammation. This dimer effectively leverages the synergistic effects of TK and monomeric glutathione, significantly enhancing the antioxidant therapeutic effect.

[0047] Therefore, on the one hand, GSH dimer can scavenge DPPH free radicals by reacting with thioketal, and on the other hand, the released reduced GSH can also effectively scavenge DPPH free radicals.

Claims

1. A glutathione dimer with active oxygen response, characterized in that: The dimer is shown in Formula I:

2. A method for preparing the glutathione dimer according to claim 1, characterized in that: The following steps are involved: Glutathione was freeze-dried, mixed with an organic solvent, and then trifluoroacetic acid was added. The mixture was stirred at room temperature overnight and precipitated with cold ether to obtain a white precipitate. The precipitate was filtered and dried to obtain a glutathione dimer with active oxygen response.

3. A method for preparing the glutathione dimer according to claim 1, characterized in that: The following steps are involved: Glutathione is freeze-dried, mixed with an organic solvent, heated, and a magnesium bromide ether solution is added to obtain a mixture; 2,2-dimethoxypropane dissolved in anhydrous acetonitrile is dripped into the mixture, stirred overnight, concentrated by rotary evaporation, precipitated with cold ether, and vacuum dried to obtain a glutathione dimer with active oxygen response.

4. The method according to claim 2 or 3, characterized in that: The glutathione is reduced glutathione, and its structure is shown in Formula II:

5. The method according to claim 2 or 3, characterized in that: The organic solvent is acetone or anhydrous acetonitrile, and the molar ratio of glutathione to the organic solvent is 1:1-5.

6. A method for preparing the glutathione dimer according to claim 1, characterized in that: The method comprises the following steps: mixing glutathione and acetone, dropping tributylphosphine, stirring and reacting at room temperature overnight, separating and collecting products by column chromatography, concentrating under reduced pressure, and vacuum drying to obtain glutathione dimers with active oxygen response.

7. The method according to claim 6, characterized in that: The glutathione is oxidized glutathione, and its structure is shown in Formula III:

8. The method according to claim 6, characterized in that: The molar ratio of glutathione to acetone is 1:1-5.

9. Use of the glutathione dimer according to claim 1 in the preparation of a drug for inhibiting oxidative stress and / or treating inflammation.

10. The application according to claim 9, characterized in that: The thioketal bond in the glutathione dimer reacts with ROS at the inflammatory site to remove ROS, and releases endogenous antioxidant tripeptide GSH to achieve the effect of synergistic antioxidant and / or inflammatory treatment.

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