A ROS-responsive endothelial cell-targeting prodrug compound, its preparation method, and its application.

Abstract

This invention discloses a ROS-responsive endothelial cell-targeting prodrug compound, its preparation method, and its applications, relating to the fields of biomedicine and medicinal chemistry. The ROS-responsive endothelial cell-targeting prodrug compound is VHP-TK-Tro. The preparation method of this prodrug compound includes the following steps: S1, reacting the ketothiol diamine derivative TK-NH2 with triphosgene to obtain an activated intermediate; S2, reacting the activated intermediate with Trolox to obtain TK-Trolox; S3, reacting TK-Trolox with the targeting peptide VHPKQHRGDWC. This prodrug compound is used in the preparation of drugs for the treatment or prevention of oxidative stress-mediated inflammatory diseases. This prodrug compound achieves active targeted drug delivery. The preparation method provided by this invention is simple, mild, and produces a high-purity product, making it easy to scale up production. This prodrug compound is expected to provide a novel, highly effective, and low-toxicity therapeutic strategy for related diseases.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and medicinal chemistry, and in particular to a ROS-responsive endothelial cell-targeting prodrug compound, its preparation method, and its application. Background Technology

[0002] Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are common critical illnesses in clinical practice, characterized by high morbidity and mortality. Its pathological features mainly include alveolar-capillary barrier disruption, pulmonary edema, inflammatory cell infiltration, and oxidative stress. Current clinical treatment primarily focuses on supportive care, lacking effective targeted interventions.

[0003] Oxidative stress plays a crucial role in the development and progression of ALI / ARDS. Excessive reactive oxygen species (ROS) can lead to a vicious cycle of endothelial cell dysfunction, mitochondrial damage, apoptosis, and inflammatory responses, thereby exacerbating lung tissue damage. Therefore, scavenging excess ROS and protecting endothelial cell function has become one of the important strategies for treating ALI.

[0004] Trolox is a water-soluble vitamin E analog with potent antioxidant activity. It can scavenge various free radicals, inhibit lipid peroxidation, and protect mitochondrial function, exhibiting good cytoprotective effects. However, Trolox lacks tissue selectivity, making it difficult to accumulate at lesion sites after systemic administration, which limits its efficacy and application prospects in the treatment of ALI.

[0005] Vascular cell adhesion molecule-1 (VCAM-1) is expressed at low levels in the resting state, but its expression is significantly upregulated under inflammatory stimulation, especially in pulmonary vascular endothelial cells, making it an important target for inflammation-related diseases. VHPKQHRGDWC is a VCAM-1 targeting peptide screened from an autophage display library. It can specifically recognize and bind to activated endothelial cells, demonstrating good potential for targeted delivery.

[0006] In recent years, ROS-responsive prodrug design has emerged as a new targeted delivery strategy. Thioketal (TK), as a ROS-sensitive linker, can be selectively cleaved in high ROS environments, enabling precise drug release at the lesion site, thereby improving efficacy and reducing systemic toxicity.

[0007] Based on the above background, this invention connects the VCAM-1 targeting peptide VHPKQHRGDWC with the antioxidant Trolox via a ROS-sensitive ketothiolate bond, constructing a prodrug molecule, VHP-TK-Tro, that exhibits endothelial cell targeting and ROS responsiveness. This molecule can be targeted and delivered to activated pulmonary vascular endothelial cells under inflammatory conditions, and releases Trolox in a local high-ROS environment, exerting multiple effects such as antioxidation, protection of mitochondrial function, inhibition of endothelial cell apoptosis, and maintenance of the lung air-blood barrier integrity, thereby achieving precise drug treatment for ALI. This invention provides a new strategy and candidate molecule for targeted therapy of acute lung injury. Summary of the Invention

[0008] In view of this, the present invention addresses the deficiencies of the existing technology, and its main objective is to provide a ROS-responsive endothelial cell-targeting prodrug compound, its preparation method, and its application. This prodrug compound utilizes the high affinity of the targeting peptide for vascular cell adhesion molecule-1, enabling it to accumulate in activated endothelial cells and achieve active targeted drug delivery. The preparation method provided by the present invention is simple in steps, mild in conditions, produces high-purity products, and is easy to scale up for production. This prodrug compound is expected to provide a new, highly effective, and low-toxicity therapeutic strategy for related diseases.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A ROS-responsive endothelial cell-targeting prodrug compound, wherein the prodrug compound is VHP-TK-Tro, formed by coupling the antioxidant Trolox with the peptide VHPKQHRGDWC, which targets vascular cell adhesion molecule-1, via a ROS-sensitive ketothiolate linker.

[0010] As a preferred embodiment, the polypeptide VHPKQHRGDWC is linked to one end of the ketothiolate bond via the thiol group of its cysteine ​​residue, and the other end of the ketothiolate bond is linked to the carboxyl group of Trolox via an ester bond or an amide bond.

[0011] A method for preparing a ROS-responsive endothelial cell-targeting prodrug compound includes the following steps: S1. The ketethiol diamine derivative TK-NH2 is reacted with triphosgene to obtain an activated intermediate; S2. The activated intermediate obtained in step S1 is reacted with Trolox to obtain the intermediate product TK-Trolox; S3. The intermediate product TK-Trolox obtained in step S2 is reacted with the target peptide VHPKQHRGDWC, and the target product VHP-TK-Tro is obtained after extraction and purification.

[0012] As a preferred embodiment, the reaction in step S1 is carried out under anhydrous dichloromethane, argon protection, and ice bath conditions.

[0013] As a preferred embodiment, the reaction in step S2 is carried out with stirring at room temperature in the presence of N,N-diisopropylethylamine.

[0014] As a preferred embodiment, the extraction and purification in step S3 are as follows: after the intermediate product TK-Trolox reacts with the targeting peptide VHPKQHRGDWC, the reaction solution is poured into 0.1 M dilute hydrochloric acid, extracted with dichloromethane, the organic phases are combined, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product is purified by preparative high performance liquid chromatography.

[0015] As a preferred embodiment, the prodrug compound is used in the preparation of a medicament for the treatment or prevention of oxidative stress-mediated inflammatory diseases.

[0016] As a preferred embodiment, the oxidative stress-mediated inflammatory disease is acute lung injury or acute respiratory distress syndrome.

[0017] A pharmaceutical composition comprising an effective amount of the ROS-responsive endothelial cell-targeting prodrug compound, and one or more pharmaceutically acceptable carriers.

[0018] Compared with existing technologies, this invention has significant advantages and beneficial effects. Specifically, as shown in the above technical solution, the prodrug compound utilizes the high affinity of the targeting peptide for vascular cell adhesion molecule-1, enabling it to accumulate in activated endothelial cells, thus achieving active targeted drug delivery and significantly increasing the drug concentration at the lesion site. Secondly, the ketithiothiolate linkage acts as a smart "switch," maintaining structural stability in a normal physiological environment and avoiding systemic drug release and the resulting side effects. When it reaches the inflammatory microenvironment, it can respond to the high concentration of reactive oxygen species (ROS) in the local environment by breaking down, precisely releasing the antioxidant Trolox, thereby promptly clearing excess ROS and inhibiting endothelial cell activation and the inflammatory cascade. This integrated "targeting-response-release" design not only enhances the efficacy of the drug but also improves its biosafety. Furthermore, the preparation method provided by this invention is simple in steps, mild in conditions, and produces high-purity products, making it easy to scale up for production. Therefore, this prodrug compound shows great clinical application potential in the preparation of drugs for treating oxidative stress-mediated inflammatory diseases such as acute lung injury / acute respiratory distress syndrome, and is expected to provide a new, highly effective, and low-toxicity treatment strategy for related diseases.

[0019] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the structures of the compound Trolox and the polypeptide VHPKQHRGDWC of the present invention. Figure 2 This is a schematic diagram of the synthetic route of the prior drug compound VHP-TK-Tro. Figure 3 This is a schematic diagram of the proton NMR spectrum (DMSO, 500 MHz) of the prior drug compound VHP-TK-Tro. Figure 4 This is a schematic diagram illustrating the reaction mechanism of the prior drug compound VHP-TK-Tro under the action of ROS (hydrogen peroxide simulation). Figure 5 This is a liquid chromatography-mass spectrometry (LC-MS) image of the prior pharmaceutical compound VHP-TK-Tro after co-incubation with hydrogen peroxide. Figure 6 This is a schematic diagram of the immunoblotting results of the VCAM-1 receptor expression changes of the present invention. Figure 7 This is a schematic diagram showing the relative uptake rates of Trolox and VHP-TK-Tro in inflamed vascular endothelial cells according to the present invention. Figure 8 This is a schematic diagram illustrating the effect of different treatments on the permeability of inflammatory vascular endothelial cell monolayers using the Transwell method of this invention. Figure 9 This is a schematic diagram showing the effect of different treatments on the level of reactive oxygen species in vascular endothelial cells and the fluorescence intensity analysis results of the CM-H2DCFDA fluorescent probe of the present invention. Figure 10 This is a schematic diagram illustrating the effect of different treatments on mitochondrial membrane potential of vascular endothelial cells using JC-1 staining according to the present invention. Figure 11 This is a schematic diagram illustrating the effect of different treatments on vascular endothelial cell apoptosis detected by Annexin V-FITC / PI double staining combined with flow cytometry according to the present invention. Figure 12 This is a schematic diagram illustrating the effects of different treatments of the present invention on the activity of superoxide dismutase and the content of malondialdehyde in vascular endothelial cells. Figure 13 This is a schematic diagram of the wet-to-dry weight ratio measurement results of lung tissue according to the present invention; Figure 14 This is a schematic diagram showing the results of the determination of total protein content in bronchoalveolar lavage fluid according to the present invention. Figure 15 This is a representative image of lung tissue stained with hematoxylin and eosin (HE) according to the present invention. Detailed Implementation

[0021] The present invention is as follows Figure 1As shown in Figure 15, a ROS-responsive endothelial cell-targeting prodrug compound is formed by coupling the antioxidant Trolox with the peptide VHPKQHRGDWC, which targets vascular cell adhesion molecule-1, via a ROS-sensitive ketothiolate linker.

[0022] The polypeptide VHPKQHRGDWC is linked to one end of the ketithiolide bond via the thiol group of its cysteine ​​residue, and the other end of the ketithiolide bond is linked to the carboxyl group of Trolox via an ester or amide bond.

[0023] A method for preparing a ROS-responsive endothelial cell-targeting prodrug compound includes the following steps: S1. The ketethiol diamine derivative TK-NH2 is reacted with triphosgene to obtain an activated intermediate; S2. The activated intermediate obtained in step S1 is reacted with Trolox to obtain the intermediate product TK-Trolox; S3. The intermediate product TK-Trolox obtained in step S2 is reacted with the target peptide VHPKQHRGDWC, and the target product VHP-TK-Tro is obtained after extraction and purification.

[0024] The reaction in step S1 is carried out under anhydrous dichloromethane, argon protection, and ice bath conditions.

[0025] The reaction in step S2 was carried out with stirring at room temperature in the presence of N,N-diisopropylethylamine.

[0026] The extraction and purification in step S3 are as follows: After the intermediate product TK-Trolox reacts with the targeting peptide VHPKQHRGDWC, the reaction solution is poured into 0.1 M dilute hydrochloric acid, extracted with dichloromethane, the organic phases are combined, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product is purified by preparative high performance liquid chromatography.

[0027] This prodrug compound is used in the preparation of drugs for the treatment or prevention of inflammatory diseases mediated by oxidative stress.

[0028] The inflammatory disease mediated by this oxidative stress is acute lung injury or acute respiratory distress syndrome.

[0029] A pharmaceutical composition comprising an effective amount of a prodrug compound and one or more pharmaceutically acceptable carriers.

[0030] Example: A ROS-responsive endothelial cell-targeting prodrug compound, its preparation method, and its application. The antioxidant Trolox was coupled to the peptide VHPKQHRGDWC, which targets vascular cell adhesion molecule-1 (VCAM-1), via a ROS-sensitive thioketal (TK) linker to form the prodrug molecule VHP-TK-Tro. This prodrug can be used for targeted therapy of acute lung injury and related oxidative stress-mediated inflammatory diseases.

[0031] Preparation method of VHP-TK-Tro Raw materials such as VHPKQHRGDWC and Trolox Figure 1 As shown, the synthetic route of VHP-TK-Tro is as follows: Figure 2 As shown. In a dry 250 mL three-necked flask, 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine (TK-NH2, 1 mmol, 194 mg) was dissolved in 20 mL of anhydrous dichloromethane. Under argon protection, the mixture was cooled in an ice bath, and triphosgene (1 mmol, 296 mg) dissolved in 10 mL of anhydrous dichloromethane was slowly added dropwise. After the addition was complete, the mixture was stirred for 1 h. Subsequently, quinoline dimethacrylate (Trolox, 1 mmol, 250 mg) and N,N-diisopropylethylamine (DIPEA, 3 mmol, 388 mg) were added, and the reaction mixture was stirred at room temperature for 4 h. After the reaction was completed by TLC monitoring, peptide VHPKQHRGDWC (0.3 mmol, approximately 408 mg) and DIPEA (1 mmol, 129 mg) were added, and the mixture was stirred at room temperature for 16 h. After the reaction was complete, the reaction solution was poured into 50 mL of dilute hydrochloric acid (0.1 M), extracted with dichloromethane (3 × 50 mL), and the organic phases were combined. After drying with anhydrous sodium sulfate and concentration under reduced pressure, the crude product was purified by preparative high-performance liquid chromatography (mobile phase: acetonitrile / water), and lyophilized to obtain a white powder product VHP-TK-Tro (187 mg, yield 35%). ¹H NMR ( Figure 3) (500 MHz, DMSO-d6) δ 12.38 (s, 1H), 12.26 (s, 1H),11.87 (s, 1H), 10.70 (d, J = 8.5 Hz, 1H), 9.17 (dd, J = 7.6, 6.6 Hz, 1H),8.95 (dd, J = 7.7, 6.7 Hz, 1H), 8.41 (d, J = 11.2 Hz, 1H), 8.34 (dd, J = 7.6,1.9 Hz, 2H), 8.20 (d, J = 11.2 Hz, 1H), 8.16-8.07 (m, 3H), 8.03 (d, J = 3.2Hz, 1H), 8.01 (d, J = 3.2 Hz, 1H), 8.02- 7.93 (m, 2H), 7.64-7.58 (m, 2H),7.33 (dt, J = 6.6, 2.1 Hz, 3H), 7.18 (d, J = 8.3 Hz, 1H), 7.13 (td, J = 7.4,1.6 Hz, 1H), 7.08 (td, J = 7.3, 1.6 Hz, 1H), 7.00 (s, 1H), 6.83 (s, 2H), 6.60(t, J = 6.3 Hz, 1H), 6.18 (t, J = 5.3 Hz, 1H), 6.12 (t, J = 8.1 Hz, 1H),6.09-6.02 (m, 2H), 5.99 -5.92 (m, 1H), 5.67 (d, J = 6.6 Hz, 1H), 4.62 (ddt, J= 18.8, 13.2, 7.0 Hz, 2H), 4.46 (ddt, J = 22.0, 11.7, 6.8 Hz, 2H), 4.31 (dt,J = 11.2, 7.0 Hz, 1H), 4.21-4.12 (m, 2H), 4.10-3.90 (m, 4H), 3.64 (td, J =8.4, 7.1 Hz, 1H), 3.53-3.27 (m, 7H), 3.21-3.00 (m, 11H), 2.93 (dt, J = 8.8,7.0 Hz, 5H), 2.92-2.85 (m, 1H), 2.87-2.82 (m, 1H), 2.85-2.76 (m, 2H), 2.58(d, J = 6.8 Hz, 2H), 2.32-2.13 (m, 13H), 2.10-1.84 (m, 6H), 1.83 -1.64 (m,5H), 1.64-1.42 (m, 17H), 1.45-1.35 (m, 2H), 1.35-1.24 (m, 1H), 0.95 (d, J =6.8 Hz, 3H), 0.90 (d, J = 6.7 Hz, 3H). .

[0032] responsive release mechanism of VHP-TK-Tro Under the action of hydrogen peroxide (H2O2), the ketithiothiol (TK) linkage in VHP-TK-Tro molecules is oxidized, leading to the breakage of the CS bond, which in turn triggers the depolymerization of the prodrug molecule, releasing the active antioxidant Trolox and the targeting peptide VHPKQHRGDWC (… Figure 4 LC-MS results showed that the chromatographic peak at retention time Rt = 6.25 min corresponded to the product of the oxidation reaction, and the molecular ion peak at m / z 249.125 in its mass spectrum was Trolox dimethacrylate, further confirming that this ROS-responsive prodrug can effectively release the drug Trolox under the action of hydrogen peroxide. Figure 5 ).

[0033] VHP-TK-Tro's endothelial cell targeting effect The expression level of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) under normal culture and after lipopolysaccharide (LPS) stimulation was detected by Western blotting. HUVECs were treated with PBS or 1 μg / mL LPS for 12 h, respectively. Total protein was extracted from the cells, separated by SDS-PAGE, and transferred to PVDF membranes. The membranes were then incubated sequentially with a rabbit polyclonal antibody against VCAM-1 (1:1000 dilution) and HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution). ECL chemiluminescence imaging was performed, and the grayscale values ​​of the bands were quantitatively analyzed using ImageJ software, with β-actin as an internal reference for correction. The results showed that compared with the PBS-treated control group, the LPS-stimulated group had a significantly increased VCAM-1 protein expression level, indicating a significant increase in VCAM-1 expression on the surface of endothelial cells under inflammatory conditions. This result provides a key molecular basis for subsequent selective drug delivery using the VCAM-1 targeting peptide VHPKQHRGDWC.

[0034] To further verify the targeted uptake capability of VHP-TK-Tro, HUVECs pre-stimulated with LPS (1 μg / mL) for 12 h were co-incubated with equimolar concentrations (10 μM) of free Trolox or VHP-TK-Tro at 37℃ and 5% CO2 for 6 h. After incubation, cells were washed three times with pre-cooled PBS to remove unbound drug, and intracellular drug was extracted with RIPA lysis buffer. The supernatant was collected by centrifugation and quantitatively detected by high-performance liquid chromatography (HPLC). The results are as follows: Figure 7 As shown, the intracellular drug content in the VHP-TK-Tro group was significantly higher than that in the free Trolox group, with a statistically significant difference (p < 0.0001). This result indicates that VHP-TK-Tro can be efficiently enriched in inflammatory-activated endothelial cells via VCAM-1-mediated endocytosis, significantly enhancing the intracellular delivery efficiency of Trolox, thus confirming that this prodrug molecule has good targeting properties to inflammatory endothelial cells.

[0035] VHP-TK-Tro reduces the permeability of inflamed vascular endothelial cells. The effects of different treatments on the permeability of human umbilical vein endothelial cells (HUVECs) monolayer were evaluated using the Transwell system. HUVECs were seeded in the upper chamber of a Transwell chamber (0.4 μm pore size) and cultured for 48 h to form a dense monolayer. The following treatments were then administered: (1) PBS control group; (2) LPS (1 μg / mL) stimulation group; (3) LPS + free Trolox group; (4) LPS + VHP-TK-Tro group. After treatment, culture medium containing FITC-labeled dextran (40 kDa, 1 mg / mL) was added to the upper chamber, and the mixture was incubated at 37°C for 30 min. The culture medium in the lower chamber was collected, and the fluorescence intensity was measured using a fluorescence spectrophotometer (excitation wavelength 490 nm, emission wavelength 520 nm) to reflect the flux of FITC-dextran through the endothelial monolayer. The results are as follows: Figure 8 As shown, compared with the PBS control group, the permeability of the endothelial cell monolayer in the LPS-stimulated group was significantly increased, as evidenced by a marked increase in FITC-dextran fluorescence intensity. Compared with the LPS group, free Trolox treatment could partially reduce the LPS-induced increase in permeability, while VHP-TK-Tro treatment more effectively inhibited the LPS-induced increase in HUVEC monolayer permeability, with its fluorescence intensity approaching that of the control group. These results indicate that VHP-TK-Tro can significantly protect endothelial barrier function and alleviate LPS-induced endothelial hyperpermeability, exhibiting a superior barrier protective effect compared to free Trolox.

[0036] VHP-TK-Tro reduces reactive oxygen species levels in inflamed vascular endothelial cells. The effects of different treatments on reactive oxygen species (ROS) levels in human umbilical vein endothelial cells (HUVECs) were detected using the CM-H2DCFDA fluorescent probe. HUVECs were seeded in culture plates and allowed to adhere overnight. After pre-stimulation with LPS (1 μg / mL) for 12 h, they were treated with PBS (control group), LPS alone, LPS + free Trolox, or LPS + VHP-TK-Tro for 24 h, respectively. After incubation, 5 μM CM-H2DCFDA probe was added, and the cells were stained at 37℃ in the dark for 30 min. After washing, the fluorescence intensity was detected using a fluorescence microplate reader (excitation wavelength 488 nm, emission wavelength 530 nm). The results are shown below. Figure 9 As shown, compared with the PBS control group, the intracellular ROS level in the LPS-stimulated group was significantly increased (mean fluorescence intensity: 31.7±4.5). Compared with the LPS group, free Trolox treatment partially reduced the ROS level (mean fluorescence intensity: 20.7±3.1), while VHP-TK-Tro treatment more significantly inhibited the LPS-induced ROS increase (mean fluorescence intensity: 10.3±0.5), and its ROS level was close to that of the PBS control group (mean fluorescence intensity: 0.5±0.3). These results indicate that VHP-TK-Tro can effectively remove excess ROS induced by LPS stimulation in endothelial cells, and its antioxidant effect is superior to that of free Trolox.

[0037] VHP-TK-Tro improves abnormal mitochondrial membrane potential in inflamed vascular endothelial cells. The effects of different treatments on the mitochondrial membrane potential (MMP) of human umbilical vein endothelial cells (HUVECs) were detected using the JC-1 fluorescent probe. HUVECs were seeded in culture plates and allowed to adhere overnight. After pre-stimulation with LPS (1 μg / mL) for 12 h, they were treated with PBS (control group), LPS alone, LPS + free Trolox, or LPS + VHP-TK-Tro for 24 h, respectively. After incubation, JC-1 working solution was added according to the kit instructions, and staining was performed at 37°C in the dark for 20 min. Immediately after washing, the changes in red and green fluorescence intensity were observed under a fluorescence microscope. When the mitochondrial membrane potential was normal, JC-1 existed in polymeric form and exhibited red fluorescence; when the membrane potential decreased, JC-1 existed in monomeric form and exhibited green fluorescence. Results are as follows: Figure 10As shown, cells in the PBS control group exhibited bright red fluorescence and weak green fluorescence; cells in the LPS-stimulated group showed significantly weakened red fluorescence and significantly enhanced green fluorescence, indicating a decrease in mitochondrial membrane potential. Compared with the LPS group, treatment with free Trolox partially restored red fluorescence, but the improvement effect was limited; while the VHP-TK-Tro treatment group showed significantly enhanced red fluorescence and weakened green fluorescence, with a significantly better effect than the free Trolox group. These results indicate that VHP-TK-Tro can effectively improve LPS-induced abnormal mitochondrial membrane potential in HUVECs and protect mitochondrial function, with a superior effect compared to free Trolox.

[0038] VHP-TK-Tro improves apoptosis in inflammatory vascular endothelial cells The effects of different treatments on apoptosis of human umbilical vein endothelial cells (HUVECs) were detected by Annexin V-FITC / PI double staining combined with flow cytometry. HUVECs were seeded in culture plates and allowed to adhere overnight. After pre-stimulation with LPS (1 μg / mL) for 12 h, cells were treated with PBS (control group), LPS alone, LPS + free Trolox, or LPS + VHP-TK-Tro for 24 h, respectively. After incubation, cells were collected, washed with pre-cooled PBS, resuspended in binding buffer, and then stained with Annexin V-FITC and propidium iodide (PI) sequentially at room temperature in the dark for 15 min. The apoptosis rate was immediately detected by flow cytometry. Results are shown below. Figure 11 As shown, the apoptosis rate in the PBS control group was approximately 5.5%; the apoptosis rate in the LPS-stimulated group significantly increased to 78.2%, indicating that LPS can induce significant apoptosis in endothelial cells. Compared with the LPS group, treatment with free Trolox partially inhibited apoptosis, reducing the apoptosis rate to 39.8%; while the apoptosis rate in the VHP-TK-Tro treatment group further decreased to 29.3%, lower than that in the free Trolox group. These results indicate that VHP-TK-Tro can effectively inhibit LPS-induced apoptosis in HUVECs, and its anti-apoptotic effect is superior to that of free Trolox.

[0039] VHP-TK-Tro improves oxidative stress damage in inflammatory vascular endothelial cells. The effects of different treatments on superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in human umbilical vein endothelial cells (HUVECs) were assessed using commercially available kits to evaluate oxidative stress levels. HUVECs were seeded in culture plates and allowed to adhere overnight. After pre-stimulation with LPS (1 μg / mL) for 12 h, they were treated with PBS (control group), LPS alone, LPS + free Trolox, or LPS + VHP-TK-Tro for 24 h, respectively. After incubation, cell lysates were collected, and the supernatant was obtained by centrifugation. SOD activity and MDA content were measured according to the kit instructions. SOD activity was measured based on its ability to inhibit the reduction of water-soluble tetrazolium salts, with absorbance detected at 450 nm. MDA content was determined using the thiobarbituric acid (TBA) method, with absorbance of the MDA-TBA adduct detected at 532 nm. Results are shown below. Figure 12 As shown, compared with the PBS control group, the LPS-stimulated group showed significantly decreased SOD activity (PBS group: 83.9±2.2 U / mL vs LPS group: 30.8±2.2 U / mL) and significantly increased MDA content (PBS group: 7.7±0.4 nmol / mL vs LPS group: 34.9±1.8 nmol / mL), indicating that LPS induced significant oxidative stress damage. Compared with the LPS group, free Trolox treatment could partially restore SOD activity (45.2±6.4 U / mL) and reduce MDA content (26.8±4.1 nmol / mL); while the VHP-TK-Tro treatment group showed significantly increased SOD activity to 69.2±7.1 U / mL and significantly decreased MDA content to 16.3±5.0 nmol / mL, with significantly better effects than the free Trolox group. These results indicate that VHP-TK-Tro can effectively alleviate LPS-induced oxidative stress in vascular endothelial cells, manifested by enhanced SOD activity and reduced generation of lipid peroxidation product MDA, and its antioxidant protective effect is superior to that of free Trolox.

[0040] The effect of VHP-TK-Tro on improving lung injury in mice with acute lung injury The wet / dry weight ratio (W / D ratio) of lung tissue was used to assess the effect of different treatments on the degree of pulmonary edema in mice with LPS-induced acute lung injury. Male Balb / c mice were randomly divided into four groups: Sham group (sham-operated control group), PBS group (model group), Trolox group (free Trolox treatment group), and VHP-TK-Tro group (prodrug treatment group of this invention). Mice in each group were infused with LPS (5 mg / kg) via tracheal instillation to establish an ALI model, while the Sham group was infused with an equal volume of PBS. 24 h after model establishment, the mice were treated with the corresponding drugs. 24 h after drug administration, the mice were sacrificed, and the right lung tissue was harvested and weighed wetly. After drying at 65℃ for 72 h to constant weight, the dry weight was measured, and the wet / dry weight ratio was calculated. Results are as follows: Figure 13 As shown, the wet-to-dryness ratio of lung tissue in the Sham group was 4.00±0.12; the wet-to-dryness ratio in the PBS model group was significantly increased to 6.16±0.12, indicating that LPS induced significant pulmonary edema. Compared with the PBS group, free Trolox treatment could partially reduce the wet-to-dryness ratio to 5.43±0.07; while the wet-to-dryness ratio in the VHP-TK-Tro treatment group was further reduced to 4.58±0.06, significantly lower than that in the free Trolox group and close to the level of the Sham group. These results indicate that VHP-TK-Tro can effectively alleviate LPS-induced pulmonary edema, and its effect is superior to that of free Trolox.

[0041] The total protein content in bronchoalveolar lavage fluid (BALF) was determined using the BCA method to assess the integrity of the alveolar-capillary barrier. Mice in each group underwent the above treatment, BALF was collected, the supernatant was collected by centrifugation, and the total protein concentration was determined according to the BCA kit instructions. Results are as follows: Figure 14 As shown, the total protein concentration in the BALF of the Sham group was 129.2 ± 5.4 μg / mL; the total protein concentration in the PBS model group was significantly increased to 448.8 ± 26.0 μg / mL, indicating that LPS caused severe damage to the alveolar-capillary barrier; compared with the PBS group, free Trolox treatment could partially reduce the total protein concentration to 327.5 ± 24.9 μg / mL; while the total protein concentration in the VHP-TK-Tro treatment group was further reduced to 242.5 ± 36.1 μg / mL, which was significantly lower than that in the free Trolox group, indicating that VHP-TK-Tro can more effectively protect the alveolar-capillary barrier and reduce protein leakage.

[0042] Hematoxylin-eosin (HE) staining was used to observe pathological changes in lung tissue. Left lung tissue was harvested from mice after sacrifice in each group. After fixation, embedding, and sectioning, HE staining was performed, and morphological changes in the lung tissue were observed under a light microscope. Results are as follows: Figure 15 As shown, the Sham group exhibited clear lung tissue structure, normal alveolar septa, and no significant inflammatory cell infiltration. The PBS model group showed severe alveolar structural damage, significantly thickened alveolar septa, and abundant inflammatory cell infiltration, exhibiting typical pathological features of acute lung injury. The free Trolox treatment group showed some relief of these pathological changes, but some alveolar structural abnormalities and inflammatory cell infiltration remained. In contrast, the VHP-TK-Tro treatment group showed significant improvement in lung tissue structure, with alveolar septa approaching normal, significantly reduced inflammatory cell infiltration, and a significantly milder degree of pathological damage compared to the free Trolox group. These results indicate that VHP-TK-Tro can significantly improve LPS-induced lung tissue pathological damage, and its protective effect is superior to that of free Trolox.

[0043] The key design feature of this invention is that the prodrug compound utilizes the high affinity of the targeting peptide for vascular cell adhesion molecule-1, enabling it to accumulate in activated endothelial cells and achieving active targeted drug delivery, significantly increasing drug concentration at the lesion site. Secondly, the ketithiothiolate linkage acts as a smart "switch," maintaining structural stability in normal physiological environments and avoiding systemic drug release and resulting side effects. Upon reaching the inflammatory microenvironment, it responds to the high concentration of reactive oxygen species (ROS) by breaking down, precisely releasing the antioxidant Trolox, thereby promptly clearing excess ROS and inhibiting endothelial cell activation and the inflammatory cascade. This integrated "targeting-response-release" design not only enhances the drug's efficacy but also improves its biosafety. Furthermore, the preparation method provided by this invention is simple, mild, and produces a high-purity product, facilitating large-scale production. Therefore, this prodrug compound demonstrates significant clinical application potential in the preparation of drugs for treating oxidative stress-mediated inflammatory diseases such as acute lung injury / acute respiratory distress syndrome, and is expected to provide a highly effective and low-toxicity new therapeutic strategy for these diseases.

[0044] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A ROS-responsive endothelial cell-targeting prodrug compound, characterized in that: The prodrug compound is VHP-TK-Tro, formed by coupling the antioxidant Trolox with the polypeptide VHPKQHRGDWC, which targets vascular cell adhesion molecule-1, via a ROS-sensitive ketothiol linkage.

2. The ROS-responsive endothelial cell-targeting prodrug compound according to claim 1, characterized in that: The polypeptide VHPKQHRGDWC is linked to one end of the ketithiolide bond via the thiol group of its cysteine ​​residue, and the other end of the ketithiolide bond is linked to the carboxyl group of Trolox via an ester bond or an amide bond.

3. A method for preparing a ROS-responsive endothelial cell-targeting prodrug compound as described in any one of claims 1-2, characterized in that: Includes the following steps: S1. The ketethiol diamine derivative TK-NH2 is reacted with triphosgene to obtain an activated intermediate; S2. The activated intermediate obtained in step S1 is reacted with Trolox to obtain the intermediate product TK-Trolox; S3. The intermediate product TK-Trolox obtained in step S2 is reacted with the target peptide VHPKQHRGDWC, and the target product VHP-TK-Tro is obtained after extraction and purification.

4. The preparation method according to claim 3, characterized in that: The reaction in step S1 is carried out under anhydrous dichloromethane, argon protection, and ice bath conditions.

5. The preparation method according to claim 3, characterized in that: The reaction in step S2 is carried out with stirring at room temperature in the presence of N,N-diisopropylethylamine.

6. The preparation method according to claim 3, characterized in that: The extraction and purification in step S3 are as follows: After the intermediate product TK-Trolox reacts with the targeting peptide VHPKQHRGDWC, the reaction solution is poured into 0.1 M dilute hydrochloric acid, extracted with dichloromethane, the organic phases are combined, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product is purified by preparative high performance liquid chromatography.

7. The application of a ROS-responsive endothelial cell-targeting prodrug compound as described in any one of claims 1-2, characterized in that: The prodrug compound is used in the preparation of a medicament for the treatment or prevention of oxidative stress-mediated inflammatory diseases.

8. The application according to claim 7, characterized in that: The inflammatory diseases mediated by oxidative stress are acute lung injury or acute respiratory distress syndrome.

9. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises an effective amount of the ROS-responsive endothelial cell-targeting prodrug compound as described in any one of claims 1-2, and one or more pharmaceutically acceptable carriers.

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